Thalidomide Analogs

ABSTRACT

Thalidomide analogs that modulate tumor necrosis factor alpha (TNFα) activity and angiogenesis are disclosed. In particularly disclosed embodiments, the thalidomide analogs are isosteric sulfur-containing analogs. Also disclosed are methods of treating a subject with the analogs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 60/504,724, filed Sep. 17, 2003, which application isincorporated by reference herein.

FIELD

The present invention relates to thalidomide analogs, methods ofsynthesizing the analogs, and methods for using the analogs to modulateangiogenesis and tumor necrosis factor alpha activities in a subject.More particularly, the invention relates to sulfur-containingthalidomide analogs and methods of making and using the same.

BACKGROUND

Thalidomide (N-α-phthalimidoglutarimide) is a glutamic acid derivativethat was introduced onto the market as a sedative hypnotic in 1956, butwas withdrawn in 1961 due to the development of severe congenitalabnormalities in babies born to mothers using it for morning sickness.Interest in the agent was reawakened after thalidomide was foundclinically effective in the treatment of erythema nodosum leprosum (ENL)and in the treatment of HIV wasting syndrome and various cancers.Mechanistic studies of its ENL activity demonstrated an anti-tumornecrosis factor alpha (anti-TNF-α) action. Specifically, thalidomideenhances the degradation of TNF-α RNA, and thereby lowers its synthesisand secretion. Further studies have defined it to be a co-stimulator ofboth CD8+ and CD4+ T cells, an inhibitor of angiogenesis via itsinhibitory actions on basic fibroblast growth factor (bFGF) and vascularendothelial growth factor (VEGF), and an inhibitor of the transcriptionfactor, NFκB.

TNF-α and family members play pivotal roles in a variety ofphysiological and pathological processes, which include cellproliferation and differentiation, apoptosis, the modulation of immuneresponses and induction of inflammation. TNF-α acts via two receptors,TNFR1 and 2. The former is expressed in all tissues and is thepredominant signaling receptor for TNF-α. The latter is primarilyexpressed on immune cells and mediates more limited biologicalresponses. The exposure of cells to TNF-α can result in activation of acaspase cascade leading to cell death via apoptosis. Indeed, major cellsurface molecules capable of initiating apoptosis are members of the TNFfamily of ligands and receptors. For example, death-inducing members ofthe TNF receptor family each contain a cytoplasmic ‘death domain’ (DD),which is a protein-protein interaction motif critical for engagingdownstream components of the signal transduction machinery.

Recently, TRAIL, the tumor necrosis factor-related apoptosis-inducingligand, has been shown to selectively induce apoptosis of tumor cells,but not most normal cells. It is indicated that TRAIL mediates thymocyteapoptosis and is important in the induction of autoimmune diseases. Moreoften, however, TNF-α receptor binding induces the activation oftranscription factors, AP-1 and NFκB, that thereafter induce genesinvolved in acute and chronic inflammatory responses. Overproduction ofTNF-α has thus been implicated in many inflammatory diseases, such asrheumatoid arthritis, graft-versus-host disease and Crohn's disease, andit additionally exacerbates ENL, septic shock, AIDS and dementiaassociated with Alzheimer's disease (AD).

A number of thalidomide analogs optimized to reduce TNF-α synthesis havebeen designed and synthesized. Primarily, these analogs includestructural modifications of the phthaloyl ring or glutarimide ring ofthalidomide. In addition, following the demonstration that theanti-angiogenic property of thalidomide is associated with itshydroxylated, open-ring metabolites, syntheses of the hydroxylated andhydrolysis metabolites as inhibitors of angiogenesis or tumor metastasishave been reported. Although extensive studies exist regarding thestructure-activity relationships between thalidomide and TNF-α, verylittle is known about the contribution of the four amide carbonyl groupsof thalidomide to its biological activity.

SUMMARY

Thalidomide analogs having angiogenesis modulating activity and TNF-αmodulating activity are disclosed. In some embodiments, the disclosedthalidomide analogs are sulfur-analogs of thalidomide, its open-ringmetabolites and its derivatives (such as its hydroxylated derivatives)in which one or more carbonyl groups are replaced by thiocarbonylgroups. For example, in some embodiments, thalidomide analogs wherein atleast one carbonyl group on the pthaloyl moiety or on the glutaramidemoiety (or its open ring form) of a thalidomide or a thalidomide analogis replaced by a thiocarbonyl group. In particular embodiments,successive replacement of the carbonyl groups in thalidomide withthiocarbonyl groups provides thiothalidomide analogs having increasedTNF-α inhibitory, activity. Surprisingly, the increase in TNF-αinhibition due to replacement of the carbonyl groups of thalidomide withthiocarbonyl groups is not associated with toxicity.

Improved methods for making thalidomide and thalidomide analogs are alsodisclosed, as are methods of converting thalidomide analogs intothiothalidomides. Due to their angiogenesis and TNF-α modulatingactivity, the disclosed thalidomide analogs, especially the disclosedthiothalidomides, can be used to treat a subject having a disease orcondition related to angiogenesis or TNF-α activity, such as a tumor orunwanted neovascularization. Furthermore, the physical and toxicologicalproperties of the disclosed thiothalidomide analogs make them suitablefor potently and safely modulating angiogenesis and TNF-α activitywithout injection, for example, by oral administration. This is incontrast to many currently available agents used for such purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the TNF-α inhibitory action of severaldisclosed thalidomide analogs in murine cells having a luciferasereporter element plus the 3′-UTR of human TNF-α relative to their actionin cells lacking the 3′-UTR.

FIG. 2 is a bar graph showing the relative angiogenic modulatingactivity of 1,3-Dioxo-2-(2-hydroxy-6-methoxypyridin-3-yl)-isoindolinehydrobromide at several concentrations.

FIG. 3 is a bar graph showing the relative angiogenic modulatingactivity of 2-(3-cyclohexenyl)-H-isoindol-1,3(2H)-dithione at severalconcentrations.

FIG. 4 is a bar graph showing the relative angiogenic modulatingactivity of 1-(2,6-Dithioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione atseveral concentrations.

FIG. 5 is a bar graph showing the relative angiogenic modulatingactivity of 3-Camphanic amino-2,6-piperidinedione at severalconcentrations.

FIG. 6 is a bar graph showing the relative angiogenic modulatingactivity of Dithiophthslimide at several concentrations.

FIG. 7 is a bar graph showing the relative angiogenic modulatingactivity of 2-(1,3-Dihydro-1-oxo-3-thioxo-2H-isoindol-2-yl)-pentanedioicacid at several concentrations.

FIG. 8 is a bar graph showing the relative angiogenic modulatingactivity of 2-(2-Oxo-6-thioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dioneat several concentrations.

FIG. 9 is a bar graph showing the relative angiogenic modulatingactivity of2,3-Dihydro-3-thioxo-2-(2,6-dithioxo-3-piperidinyl)-1H-isoindol-1-one atseveral concentrations.

FIG. 10 is a bar graph showing the relative angiogenic modulatingactivity of 2-Acetoxy-N-(2,6-dioxopiperidin-3-yl)benzamide at severalconcentrations.

FIG. 11 is a bar graph showing the relative angiogenic modulatingactivity of 1,3-Dioxo-2-(2,6-dimethoxypyridin-3-yl)-isoindoline atseveral concentrations.

DETAILED DESCRIPTION OF PARTICULARLY DISCLOSED EMBODIMENTS I.Abbreviations

TNF-α—tumor necrosis factor alpha

CDI—carboxyamidotriazole

ARE—adenylate/uridylate (AI)-rich element

UTR—untranslated region

THF—tetrahydrofuran

NMR—nuclear magnetic resonance

LR—Lawesson's Reagent

II Terms

In order to facilitate an understanding of the embodiments presented,the following explanations are provided.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. The term “comprises” means “includes.” Also, “comprising A orB” means including A or B, or A and B, unless the context clearlyindicates otherwise. It is to be further understood that all molecularweight or molecular mass values given for compounds are approximate, andare provided for description. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of this disclosure, suitable methods and materials are describedbelow. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The term “subject” refers to animals, including mammals (for example,humans and veterinary animals such as dogs, cats, pigs, horses, sheep,and cattle).

An “R-group” or “substituent” refers to a single atom (for example, ahalogen atom) or a group of two or more atoms that are covalently bondedto each other, which are covalently bonded to an atom or atoms in amolecule to satisfy the valency requirements of the atom or atoms of themolecule, typically in place of a hydrogen atom. Examples ofR-groups/substituents include alkyl groups, hydroxyl groups, alkoxygroups, acyloxy groups, mercapto groups, and aryl groups.

“Substituted” or “substitution” refer to replacement of a hydrogen atomof a molecule or an R-group with one or more additional R-groups such ashalogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy,mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino,alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl,piperazin-1-yl, nitro, sulfato or other R-groups.

“Alkyl” refers to a cyclic, branched, or straight chain group containingonly carbon and hydrogen, and unless otherwise mentioned typicallycontains one to twelve carbon atoms. This term is further exemplified bygroups such as methyl, ethyl, n-propyl, isobutyl, t-butyl, pentyl,pivalyl, heptyl, adamantyl, and cyclopentyl. Alkyl groups can either beunsubstituted or substituted. “Lower alkyl” groups are those thatcontain one to six carbon atoms.

“Acyl” refers to a group having the structure RCO—, where R may bealkyl, or substituted alkyl. “Lower acyl” groups are those that containone to six carbon atoms.

“Acyloxy” refers to a group having the structure RCOO—, where R may bealkyl or substituted alkyl. “Lower acyloxy” groups contain one to sixcarbon atoms.

“Alkenyl” refers to a cyclic, branched or straight chain groupcontaining only carbon and hydrogen, and unless otherwise mentionedtypically contains one to twelve carbon atoms, and contains one or moredouble bonds that may or may not be conjugated. Alkenyl groups may beunsubstituted or substituted. “Lower alkenyl” groups contain one to sixcarbon atoms.

“Alkynyl” refers to a cyclic, branched or straight chain groupcontaining only carbon and hydrogen, and unless otherwise mentionedtypically contains one to twelve carbon atoms, and contains one or moretriple bonds. Alkynyl groups may be unsubstituted or substituted. “Loweralkynyl” groups are those that contain one to six carbon atoms.

“Alkoxy” refers to a group having the structure R—O—, where R may bealkyl or substituted alkyl. Examples of alkoxy groups include methoxy,ethoxy, propoxy and butoxy groups. “Lower alkoxy” groups are those thatcontain one to six carbon atoms.

The term “halogen” refers to fluoro, bromo, chloro and iodosubstituents.

“Aryl” refers to a monovalent unsaturated aromatic carbocyclic grouphaving a single ring (e.g., phenyl) or multiple condensed rings (e.g.,naphthyl or anthryl), which can optionally be unsubstituted orsubstituted.

The term “amino” refers to an R-group having the structure —NH₂, whichcan be optionally substituted with, for example, lower alkyl groups, toyield an amino group having the general structure —NHR or —NR₂.

“Nitro” refers to an R-group having the structure —NO₂.

The term “aliphatic” as applied to cyclic groups refers to ringstructures in which any double bonds that are present in the ring arenot conjugated around the entire ring structure.

The term “aromatic” as applied to cyclic groups refers to ringstructures which contain double bonds that are conjugated around theentire ring structure, possibly through a heteroatom such as an oxygenatom or a nitrogen atom. Aryl groups, pyridyl groups and furan groupsare examples of aromatic groups. The conjugated system of an aromaticgroup contains a characteristic number of electrons, for example, 6 or10 electrons that occupy the electronic orbitals making up theconjugated system, which are typically un-hybridized p-orbitals.

“Pharmaceutical compositions” are compositions that include an amount(for example, a unit dosage) of one or more of the disclosed compoundstogether with one or more non-toxic pharmaceutically acceptableexcipients, including carriers, diluents, and/or adjuvants, andoptionally other biologically active ingredients. Such pharmaceuticalcompositions can be prepared by standard pharmaceutical formulationtechniques such as those disclosed in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. (19th Edition).

A “therapeutically effective amount” of the disclosed compounds is adosage of the compound that is sufficient to achieve a desiredtherapeutic effect, such as inhibition of angiogenesis or an anti-tumoror anti-metastatic effect, or inhibition of TNF-α activity. In someexamples, a therapeutically effective amount is an amount sufficient toachieve tissue concentrations at the site of action that are similar tothose that are shown to modulate angiogenesis or TNF-α activity intissue culture, in vitro, or in vivo. For example, a therapeuticallyeffective amount of a compound may be such that the subject receives adosage of about 0.1 μg/kg body weight/day to about 1000 mg/kg bodyweight/day, for example, a dosage of about 1 μg/kg body weight/day toabout 1000 μg/kg body weight/day, such as a dosage of about 5 μg/kg bodyweight/day to about 500 μg/kg body weight/day.

The term “stereoisomer” refers to a molecule that is an enatiomer,diasteromer or geometric isomer of a molecule. Stereoisomers, unlikestructural isomers, do not differ with respect to the number and typesof atoms in the molecule's structure but with respect to the spatialarrangement of the molecule's atoms. Examples of stereoisomers includethe (+) and (−) forms of optically active molecules.

The term “modulate” refers to the ability of a disclosed compound toalter the amount, degree, or rate of a biological function, theprogression of a disease, or amelioration of a condition. For example,modulating can refer to the ability of a compound to elicit an increaseor decrease in angiogenesis, to inhibit TNF-α activity, or to inhibittumor metastasis or tumorigenesis.

The term “angiogenic activity” refers to the ability of a disclosedcompound or a particular concentration of a disclosed compound tostimulate angiogenesis. Angiogenic activity may be detected in vivo orin vitro. Angiogenic compounds or angiogenic concentrations of disclosedcompounds stimulate angiogenesis, and such compounds and/orconcentrations may be readily identified by those of ordinary skill inthe art, using, for example, the methods described in the Examples thatfollow.

The term “anti-angiogenic activity” refers to the ability of a compoundor a particular concentration of a disclosed compound to inhibitangiogenesis. Anti-angiogenic activity may be detected in vivo or invitro. Anti-angiogenic or anti-angiogenic concentrations of disclosedcompounds inhibit angiogenesis, and such compounds and/or concentrationsmay be readily identified by those of ordinary skill in the art, using,for example, the methods described in the Examples that follow.

III. Overview of Particularly Disclosed Embodiments

Disclosed are thalidomide analogs that modulate TNF-α activity and/orangiogenesis, and as such can be used to treat a wide variety ofpathological conditions that are linked to angiogenesis and/or TNF-αactivity. Pharmaceutically acceptable salts, stereoisomers, andmetabolites of all of the disclosed compounds also are contemplated. Insome embodiments, the thalidomide analogs are thiotlialidomidederivatives in which carbonyl groups in correspondingnon-sulfur-containing thalidomide derivatives are replaced by one ormore thiocarbonyl groups.

In the structures that follow, all valency requirements are understoodto be satisfied. Thus, for example, carbon atoms have four bonds toother atoms, even if all such bonds are not shown. As is understood bythose of ordinary skill in the art, where all four bonds to a carbonatom are not shown, additional bonds to hydrogen atoms are implied.Further substitution of such implied hydrogen atoms is possible.

In other embodiments, the disclosed compounds include compounds havingthe chemical formula:

wherein X and Y are independently CH₂, oxygen or sulfur, and at leastone of X and Y is sulfur if R₁ does not include a sulfur atom; each ofR₂-R₅ are independently hydrogen, hydroxyl, acyl, substituted acyl,acyloxy, substituted acyloxy, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, amino, substituted amino, halogen, nitroor linked to form a five- or six-membered, unsubstituted or substituted,aliphatic, aromatic or heterocyclic ring, for example, hydrogen, loweralkyl, acyloxy, halogen, hydroxyl, amino or nitro such as hydrogen,acyloxy or hydroxyl; and R₁ is an unsubstituted or substituted,aliphatic or aromatic heterocyclic ring, an unsubstituted or substitutedcycloalkenyl ring, or

wherein W and Z are each independently oxygen or sulfur, R₆ and R₇ areeach independently hydroxyl, alkoxy or substituted alkoxy, and each ofR₈-R₁₂ are independently hydrogen, hydroxyl, acyl, substituted acyl,acyloxy, substituted acyloxy, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, amino, substituted amino, halogen ornitro, for example, hydrogen, lower alkyl, acyloxy, halogen, hydroxyl,amino or nitro such as hydrogen, acyloxy or hydroxyl.

In particular embodiments, R₁ is

wherein W and Z are each independently oxygen or sulfur, R₁₃ and R₁₄ areeach independently hydrogen, alkyl or substituted alkyl; R₂₀ ishydrogen, hydroxyl, alkyl or substituted alkyl such as aryl substitutedalkyl; and R₁₅-R₁₉ are each independently hydrogen, hydroxyl, acyl,substituted acyl, acyloxy, substituted acyloxy, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, aryl, substituted aryl, amino, substitutedamino, halogen or nitro, for example, hydrogen, lower alkyl, acyloxy,halogen, hydroxyl, amino or nitro such as hydrogen, acyloxy or hydroxyl.In some embodiments, at least one of R₂, R₃, R₄, R₅, R₈, R₉, R₁₀, R₁₁,R₁₅, R₁₆, R₁₇, R₁₈ and R₁₉ is hydroxyl. In other embodiments, at leastone of X, Y, W and Z is sulfur, at least two of X, Y, W and Z aresulfur, or at least three of X, Y, W and Z are sulfur. For example, inmore particular embodiments, X or Y is sulfur, and both W and Z areoxygen if present; both X and Y are sulfur and both W and Z are oxygenif present; X and Y are both oxygen and W or Z is sulfur if present;both X and Y are sulfur and W or Z is sulfur if present; or X or Y aresulfur and both W and Z are sulfur if present. Alternatively, where Wand Z are present the following are possible: X═S, Y═O, W═O, Z═O; X═O,Y═S, W═O, Z═O; X═O, Y═O, W═S, Z═O; X═O, Y═O, W═O, Z═S; X═S, Y═S, W═O,Z═O; X═S, Y═O, W═S, Z═O; X═S, Y═O, W═O, Z═S; X═O, Y═O, W═S, Z═S; X═O,Y═S, W═O, Z═S; Y═S, W═S, Z═S; X═S, Y═S, W═S, Z═O; X═S, Y═S, W═O, Z═S;X═S, Y═O, W═S, Z═S; Y═S, W═S, Z═S; or X═S, Y═S, W═S, Z═S. In otherparticular embodiments X═S and Y═CH₂.

In more particular embodiments, the disclosed compounds have the formula

wherein X, Y, W and Z are independently sulfur or oxygen and at leastone of X, Y, W and Z is sulfur, and R₂-R₁₂ are as before. For example,in more particular embodiments, X or Y is sulfur, and both W and Z areoxygen if present; both X and Y are sulfur and both W and Z are oxygenif present; X and Y are both oxygen and W or Z is sulfur if present;both X and Y are sulfur and W or Z is sulfur if present; or X or Y aresulfur and both W and Z are sulfur if present. Alternatively, where Wand Z are present the following are possible: X═S, Y═O, W═O, Z═O; X═O,Y═S, W═O, Z═O; X═O, Y═O, W═S, Z═O; X═O, Y═O, W═O, Z═S; X═S, Y═S, W═O,Z═O; X═S, Y═O, W═S, Z═O; X═S, Y═O, W═O, Z═S; X═O, Y═O, W═S, Z═S; X═O,Y═S, W═O, Z═S; X═O, Y═S, W═S, Z═O; X═S, Y═S, W═S, Z═O; X═S, Y═S, W═O,Z═S; X═S, Y═O, W═S, Z═S; X═O, Y═S, W═S, Z═S; or X═S, Y═S, W═S, Z═S. Inmore particular embodiments, at least one of R₂-R₅ and R₈-R₁₁ ishydroxyl. Specific examples of such compounds include:

In other more particular embodiments, the disclosed compounds have thechemical formula:

wherein W, X, Y and Z each are independently sulfur or oxygen and atleast one of W, X, Y and Z is sulfur; and R₂-R₅ and R₁₅-R₂₀ are asbefore. For example, in more particular embodiments, X or Y is sulfur,and both W and Z are oxygen; both X and Y are sulfur and both W and Zare oxygen; X and Y are both oxygen and W or Z is sulfur; both X and Yare sulfur and W or Z is sulfur; or X or Y are sulfur and both W and Zare sulfur. Alternatively, the following are possible: X═S, Y═O, W═O,Z═O; O X═O, Y═S, Z═O; O X═O, Yom, W═S, Z═O; O X═O, O Y═O, O W═O, O Z═S;X═S, Y═S, W═O, Z═O; X═S, Y═O, W═S, Z═O; X═S, Y═O, W═O, O Z═S; X═O, Y═O,W═S, Z═S; X═O, Y═S, W═O, Z═S; X═O, Y═S, W═S, Z═O; X═S, Y═S, W═S, Z═O;X═S, Y═S, W═O, Z═S; X═S, Y═O, W═S, Z═S; X═O, Y═S, W═S, Z═S; or X═S, Y═S,W═S, Z═S. In more particular embodiments, at least one of R₂-R₅ andR₁₅-R₁₉ is hydroxyl. Specific examples of such compounds include:

The disclosed compounds also include compounds having the formula

wherein T and V are independently oxygen or sulfur, R₂₁-R₂₅ areindependently hydrogen, hydroxyl, acyl, substituted acyl, acyloxy,substituted acyloxy, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, aryl,substituted aryl, amino, substituted amino, halogen or nitro, forexample, hydrogen, lower alkyl, acyloxy, halogen, hydroxyl, amino ornitro such as hydrogen, acyloxy or hydroxyl; and R₂₆ is

wherein W, Z and R₁₃-R₂₀ are as before. For example, in more particularembodiments, T or V is sulfur, and both W and Z are oxygen if present;both T and V are sulfur and both W and Z are oxygen if present; T and Vare both oxygen and W or Z is sulfur if present; both T and V are sulfurand W or Z is sulfur if present; or T or V are sulfur and both W and Zare sulfur if present. Alternatively, where W and Z are present thefollowing are possible: T═O, V═O, W═O, Z═O; T═S, W═O, Z═O; T═O, V═S,W═O, Z═O; T═O, V═O, W═S, Z═O; T═O, V═O, W═O, Z═S; T═S, V═S, W═O, Z═O;T═S, V═O, W═S, Z═O; T═S, V═O, W═O, Z═S; T═O, V═O, W═S, Z═S; T═O, V═S,W═O, Z═S; T═O, V═S, W═S, Z═O; T═S, V═S, W═S, Z═O; T═S, V═S, W═O, Z═S;T═S, V═O, W═S, Z═S; T═O, V═S, W═S, Z═S; or T═S, V═S, W═S, Z═S. In someembodiments, at least one of R₁₅-R₁₉ and R₂₂-R₂₆ is hydroxyl.

Still further, the disclosed compounds include compounds having theformula

wherein X, Y are each independently oxygen or sulfur; W, X and R₁₅-R₂₀are as before; R₂₇-R₃₃ are each independently hydrogen, hydroxyl, acyl,substituted acyl, acyloxy, substituted acyloxy, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, aryl, substituted aryl, amino, substitutedamino, halogen or nitro, for example, hydrogen, lower alkyl, acyloxy,halogen, hydroxyl, amino or nitro such as hydrogen, acyloxy or hydroxyl;and R₃₄ is hydrogen, alkyl or substituted alkyl. For example, in moreparticular embodiments, X or Y is sulfur, and both W and Z are oxygen;both X and Y are sulfur and both W and Z are oxygen; X and Y are bothoxygen and W or Z is sulfur; both X and Y are sulfur and W or Z issulfur; or X or Y are sulfur and both W and Z are sulfur. Alternatively,the following are possible: X═O, Y═O, W═O, Z═O; X═S, Y═O; W═O, Z═O; X═O,Y═S, W═O, Z═O; X═O, Y═O, W═S, Z═O; X═O, W═O, Z═S; X═S, Y═S, W═O, Z═O;X═S, Y═O, W═S, Z═O; X═S, Y═O, Z═S; X═O, Y═O, W═S, Z═S; X═O, Y═S, W═O,Z═S; X═O, Y═S, W═S, Z═O; X═S, Y═S, W═S, Z═O; X═S, Y═S, W═O, Z═S; X═S,Y═O, W═S, Z═S; X═O, Y═S, W═S, Z═S; or X═S, Y═S, W═S, Z═S.

In addition, the disclosed compounds include compounds having theformula

wherein X and Y are each independently oxygen or sulfur; W, Z, R₁₅-R₂₀and R₃₄ are as before, R₃₅ is alkyl or substituted alkyl, and R₃₆-R₃₉are each independently hydrogen, hydroxyl, acyl, substituted acyl,acyloxy, substituted acyloxy, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, amino, substituted amino, halogen ornitro, for example, hydrogen, lower alkyl, acyloxy, halogen, hydroxyl,amino or nitro such as hydrogen, acyloxy or hydroxyl. For example, inmore particular embodiments, X or Y is sulfur, and both W and Z areoxygen; both X and Y are sulfur and both W and Z are oxygen; X and Y areboth oxygen and W or Z is sulfur; both X and Y are sulfur and W or Z issulfur; or X or Y are sulfur and both W and Z are sulfur. Alternatively,the following are possible: X═O, Y═O, W═O, Z═O; X═S, Y═O, W═O, Z═O; X═O,Y═S, W═O, Z═O; X═O, Y═O, W═S, Z═O; X═O, Y═O, W═O, Z═S; X═S, Y═S, W═O,Z═O; X═S, Y═O, W═S, Z═O; X═S, Y═O, W═O, Z═S; X═O, Y═O, W═S, Z═S; X═O,Y═S, W═O, Z═S; X═O, Y═S, W═S, Z═O; X═S, Y═S, W═S, Z═O; X═S, Y═S, W═O,Z═S; X═S, Y═O, W═S, Z═S; X═O, Y═S, W═S, Z═S; or X═S, Y═S, W═S, Z═S.

Other embodiments include compounds having the formula

wherein X and Y each are independently oxygen or sulfur; W, Z andR₁₅-R₂₀ are as before; and R₄₀-R₄₅ are each independently hydrogen,hydroxyl, acyl, substituted acyl, acyloxy, substituted acyloxy, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, aryl, substituted aryl, amino,substituted amino, halogen or nitro, for example, hydrogen, lower alkyl,acyloxy, halogen, hydroxyl, amino or nitro such as hydrogen, acyloxy orhydroxyl. For example, in more particular embodiments, X or Y is sulfur,and both W and Z are oxygen; both X and Y are sulfur and both W and Zare oxygen; X and Y are both oxygen and W or Z is sulfur; both X and Yare sulfur and W or Z is sulfur; or X or Y are sulfur and both W and Zare sulfur. Alternatively, the following are possible: X═O, Y═O, W═O,Z═O; X═S, Y═O, W═O, Z═O; X═O, Y═S, W═O, Z═O; X═O, Y═O, W═S, Z═O; X═O,Y═O, W═O, Z═S; X═S, Y═S, W═O, Z═O; X═S, W═S, Z═O; X═S, Y═O, W═O, Z═S;X═O, Y═O, O W═S, Z═S; X═O, Y═S, W═O, Z═S; Y═S, W═S, Z═O; X═S, Y═S, W═S,Z═O; X═S, Y═S, W═O, Z═S; X═S, Y═O, W═S, S; Y═S, W═S, Z═S; or X═S, Y═S,W═S, Z═S.

The disclosed compounds further include compounds having the formula

wherein X, Y, W and Z are independently oxygen or sulfur, and R₂-R₅ andR₁₃-R₁₆ are as before. For example, in more particular embodiments, X orY is sulfur, and both W and Z are oxygen; both X and Y are sulfur andboth W and Z are oxygen; X and Y are both oxygen and W or Z is sulfur;both X and Y are sulfur and W or Z is sulfur; or X or Y are sulfur andboth W and Z are sulfur. Alternatively, the following are possible: X═O,Y═O, W═O, Z═O; X═S, Y═O, W═O, Z═O; X═O, Y═S, W═O, Z═O; X═O, Y═O, W═S,Z═O; X═O, Y═O, W═O, Z═S; X═S, Y═S, W═O, Z═O; X═S, Y═O, W═S, Z═O; X═S,Y═O, W═O, X═O, Y═O, W═S, Z═S; X═O, Y═S, W═O, Z═S; X═O, Y═S, W═S, Z═O;X═S, Y═S, W═S, Z═O; X═S, Y═S, W═O, Z═S; X═S, Y═O, W═S, Z═S; X═O, Y═S,Z═S; or X═S, Y═S, W═S, Z═S.

Also disclosed is a thalidomide analog compound having the formula

wherein X, Y and Z are independently oxygen or sulfur, and R₂-R₅,R₁₅-R₂₀ and R₃₄ are as before. For example, in more particularembodiments, X or Y is sulfur, and Z is oxygen; both X and Y are sulfurand Z is oxygen; X and Y are both oxygen and Z is sulfur. Alternatively,the following are possible: X═O, Y═O, Z═O; X═S, Z═O; X═O, Y═S, Z═O; X═O,Y═O, Z═S; X═S, Y═S, Z═O; X═S, Y═O, Z═S; X═O, Y═S, Z═S; or X═S, Y═S, Z═S.

Also disclosed is a thalidomide analog compound having the formula

wherein X and Y are independently oxygen or sulfur, and R₁, R₂, R₄ andR₅ are as before. For example, in particular embodiments, R₁ is

wherein W, Z, and R₁₃-R₂₀ are as before. For example, in more particularembodiments, X or Y is sulfur, and both W and Z are oxygen if present;both X and Y are sulfur and both W and Z are oxygen if present; X and Yare both oxygen and W or Z is sulfur if present; both X and Y are sulfurand W or Z is sulfur if present; or X or Y are sulfur and both W and Zare sulfur if present. Alternatively, where W and Z are present thefollowing are possible: X═O, Y═O, W═O, Z═O; X═S, Y═O, W═O, Z═O; X═O,Y═S, W═O, Z═O; X═O, Y═O, W═S, Z═O; X═O, Y═O, W═O, Z═S; X═S, Y═S, W═O,Z═O; X═S, Y═O, W═S, Z═O; X═S, Y═O, W═O, Z═S; X═O, Y═O, W═S, Z═S; Y═S,W═O, Z═S; X═O, Y═S, W═S, Z═O; X═S, Y═S, W═S, X═S, Y═S, W═O, Z═S; X═S,Y═O, W═S, Z═S; X═O, Y═S, W═S, Z═S; or X═S, Y═S, W═S, Z═S. In moreparticular embodiments, the compound has the formula:

wherein X, Y are independently oxygen or sulfur, and W, Z, R₂, R₄, R₅,and R₁₃-R₁₆ are as before. For example, in more particular embodiments,X or Y is sulfur, and both W and Z are oxygen; both X and Y are sulfurand both W and Z are oxygen; X and Y are both oxygen and W or Z issulfur; both X and Y are sulfur and W or Z is sulfur; or X or Y aresulfur and both W and Z are sulfur. Alternatively, the following arepossible: X═O, Y═O, W═O, Z═O; X═S, Y═O, W═O, Z═O; O X═O, Y═S, W═O, Z═O;X═O, Y═O, W═S, Z═O; X═O, Y═O, W═O, Z═S; X═S, Y═S, W═O, Z═O; X═S, Y═O,W═S, Z═O; X═S, Y═O, W═O, Z═S; X═O, Y═O, W═S, Z═S; X═O, Y═S, W═O, Z═S;X═O, Y═S, W═S, Z═O; X═S, Y═S, W═S, Z═O; X═S, Y═S, W═O, Z═S; X═S, Y═O,W═S, Z═S; X═O, Y═S, W═S, Z═S; or X═S, Y═S, W═S, Z═S. In even moreparticular embodiments, at least one of R₂, R₄, R₅, R₁₅ and R₁₆ ishydroxyl.

Also disclosed is a compound having the formula:

wherein G and D are each independently oxygen or sulfur, R₂-R₅ are asbefore, and R₄₆ is

wherein W, Z and R₁₃-R₂₀ are as before. For example, in particularembodiments, G or D is sulfur, and both W and Z are oxygen; both G and Dare sulfur and both W and Z are oxygen; G and D are both oxygen and W orZ is sulfur; both G and D are sulfur and W or Z is sulfur; or G or D aresulfur and both W and Z are sulfur. Alternatively, the following arepossible: G═O, D═O, W═O, Z═O; G═S, D═O, W═O, Z═O; G═O, D═S, W═O, Z═O;G═O, D═O, W═S, Z═O; G═O, D═O, W═O, Z═S; G═S, D═S, W═O, Z═O; G═S, D═O,W═S, Z═O; G═S, D═O, W═O, Z═S; G═O, D═O, W═S, Z═S; G═O, D═S, W═O, Z═S;G═O, D═S, W═S, Z═O; G═S, D═S, W═S, Z═O; G═S, D═S, W═O, Z═S; G═S, D═O,W═S, Z═S; G═O, D═S, W═S, Z═S; or G═S, D═S, W═S,

A method for modulating TNF-α activity in a subject also is disclosed.The method includes administering to the subject a therapeuticallyeffective amount of one or more of any of the compounds disclosed above,or a compound having the formula:

where X and Y are independently oxygen or sulfur; W, Z, R₁₅-R₂₀ are asbefore; and R₄₇-R₅₂ are each independently hydrogen, hydroxyl, acyl,substituted acyl, acyloxy, substituted acyloxy, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, aryl, substituted aryl, amino, substitutedamino, halogen or nitro, for example, hydrogen, lower alkyl, acyloxy,halogen, hydroxyl, amino or nitro such as hydrogen, acyloxy or hydroxyl;

or a compound having the formula

wherein n=1-5; X is oxygen or sulfur, and R₂-R₅ and R₁₅-R₁₉ are asbefore; or a compound having the formula:

wherein each of X and Y are independently oxygen or sulfur, n=1-5, andR₂-R₅ are as before;

or a compound having the formula:

wherein R₅₃ and R₅₄ are independently hydrogen, hydroxyl, acyl,substituted acyl, acyloxy, substituted acyloxy, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, aryl, substituted aryl, amino, substitutedamino, halogen or nitro, for example, hydrogen, lower alkyl, acyloxy,halogen, hydroxyl, amino or nitro such as hydrogen, acyloxy or hydroxyl;and R₅₅ is hydrogen, alkyl, or substituted alkyl; or a compound havingthe formula:

wherein R₂-R₅ are as before and R₅₆ is hydrogen, alkyl or substitutedalkyl;

or pharmaceutically acceptable salts or stereoisomers thereof.

Novel thio-substituted analogs having the structures described withrespect to the method above also are contemplated. For example, in moreparticular embodiments, X or Y is sulfur, and both W and Z are oxygen ifpresent; both X and Y are sulfur and both W and Z are oxygen if present;X and Y are both oxygen and W or Z is sulfur if present; both X and Yare sulfur and W or Z is sulfur if present; or X or Y are sulfur andboth W and Z are sulfur if present. Alternatively, if W and Z arepresent, the following are possible: X═O, Y═O, W═O, Z═O; X═S, Y═O, W═O,Z═O; X═O, Y═S, W═O, Z═O; X═O, Y═O, W═S, Z═O; X═O, Y═O, O W═O, Z═S; X═S,Y═S, WD, Z═O; X═S, Y═O, W═S, Z═O; X═S, Y═O, W═O, Z═S; X═O, Y═O, W═S,Z═S; X═O, Y═S, W═O, Z═S; X═O, Y═S, W═S, Z═O; X═S, Y═S, W═S, Z═O; X═S,Y═S, W═O, Z═S; X═S, Y═O, W═S, Z═S; X═O, Y═S, W═S, Z═S; or X═S, Y═S, W═S,Z═S.

Particularly disclosed compounds and compounds that can be used in thedisclosed methods include one or more compounds having the followingstructures:

Still further, a method for modulating angiogenesis in a subject isdisclosed. The method includes administering to the subject atherapeutically effective amount of one or more of any of the disclosedcompounds. Examples of compounds useful for the method are shown above.In some embodiments, where an anti-angiogenic compound or ananti-angiogenic concentration of a compound is utilized, thetherapeutically effective amount of the compound can be administered toa subject with a tumor to achieve an anti-tumor effect, such asinhibition of tumorigenesis or tumor metastasis. In other embodiments,the therapeutically effective amount of the compound is administered toa subject with a pathological angiogenesis. Alternatively, wherestimulation of angiogenesis is desired an angiogenic compound or anangiogenic concentration of a compound is administered to a subject tostimulate angiogenesis.

As angiogenesis inhibitors, the disclosed compounds are useful in thetreatment of both primary and metastatic solid tumors, includingcarcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx,esophagus, stomach, pancreas, liver, gallbladder and bile ducts, smallintestine, urinary tract (including kidney, bladder and urothelium),female genital tract, (including cervix, uterus, and ovaries as well aschoriocarcinoma and gestational trophoblastic disease), male genitaltract (including prostate, seminal vesicles, testes and germ celltumors), endocrine glands (including the thyroid, adrenal, and pituitaryglands), and skin, as well as hemangiomas, melanomas, sarcomas(including those arising from bone and soft tissues as well as Kaposi'ssarcoma) and tumors of the brain, nerves, eyes, and meninges (includingastrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,neuroblastomas, Schwannomas, and meningiomas). Such compounds may alsobe useful in treating solid tumors arising from hematopoieticmalignancies such as leukemias (i.e. chloromas, plasmacytomas and theplaques and tumors of mycosis fungoides and cutaneous T-celllymphoma/leukemia) as well as in the treatment of lymphomas (bothHodgkin's and non-Hodgkin's lymphomas). In addition, these compounds maybe useful in the prevention of metastases from the tumors describedabove either when used alone or in combination with radiotherapy and/orother chemotherapeutic agents.

Further uses of disclosed anti-angiogenic compounds/concentrationsinclude the treatment and prophylaxis of autoimmune diseases such asrheumatoid, immune and degenerative arthritis. Such compounds can alsobe used to treat a pathological (i.e. abnormal, harmful or undesired)angiogenesis, for example, various ocular diseases such as diabeticretinopathy, retinopathy of prematurity, corneal graft rejection,retrolental fibroplasia, neovascular glaucoma, rubeosis, retinalneovascularization due to macular degeneration, hypoxia, angiogenesis inthe eye associated with infection or surgical intervention, and otherabnormal neovascularization conditions of the eye; skin diseases such aspsoriasis; blood vessel diseases such as hemagiomas, and capillaryproliferation within atherosclerotic plaques; Osler-Webber Syndrome;myocardial angiogenesis; plaque neovascularization; telangiectasia;hemophiliac joints; angiofibroma; and wound granulation. Other usesinclude the treatment of diseases characterized by excessive or abnormalstimulation of endothelial cells, including but not limited tointestinal adhesions, Crohn's disease, atherosclerosis, scleroderma, andhypertrophic scars, such as keloids. Another use is as a birth controlagent, by inhibiting ovulation and establishment of the placenta. Thedisclosed compounds are also useful in the treatment of diseases thathave angiogenesis as a pathologic consequence such as cat scratchdisease (Rochele minalia quintosa) and ulcers (Helicobacter pylori). Thedisclosed compounds are also useful to reduce bleeding by administrationprior to -surgery, especially for the treatment of resectable tumors.

Angiogenic compounds or angiogenic concentrations of disclosed compoundcan be used can be used to treat a variety of conditions that wouldbenefit from stimulation of angiogenesis, stimulation of vasculogenesis,increased blood flow, and/or increased vascularity. Particular examplesof conditions and diseases amenable to treatment using disclosedangiogenic compounds, or angiogenic concentrations of disclosedcompounds, include any condition associated with an obstruction of ablood vessel, such as obstruction of an artery, vein, or of a capillarysystem. Specific examples of such conditions or disease include, but arenot necessarily limited to, coronary occlusive disease, carotidocclusive disease, arterial occlusive disease, peripheral arterialdisease, atherosclerosis, myointimal hyperplasia (such as due tovascular surgery or balloon angioplasty or vascular stenting),thromboangiitis obliterans, thrombotic disorders, vasculitis, and thelike. Examples of conditions or diseases that may be prevented using thedisclosed angiogenic compounds/concentrations include, but are notlimited to, heart attack (myocardial infarction) or other vasculardeath, stroke, death or loss of limbs associated with decreased bloodflow, and the like. Other therapeutic uses for angiogenesis stimulationaccording to the disclosure include, but are not necessarily limited toaccelerating healing of wounds or ulcers; improving the vascularizationof skin grafts or reattached limbs so as to preserve their function andviability; improving the healing of surgical anastomoses(such as inre-connecting portions of the bowel after gastrointestinal surgery); andimproving the growth of skin or hair.

Yet further, a method for inhibiting TNF-α activity in a subject usingthe disclosed compounds is provided. The method includes administering atherapeutically effective amount of a disclosed compound to a subject toachieve a TNF-α inhibitory effect. The disclosed compounds having TNF-αinhibitory effects are useful for treating many inflammatory,infectious, immunological, and malignant diseases. These include but arenot limited to septic shock, sepsis, endotoxic shock, hemodynamic shockand sepsis syndrome, post ischemic reperfusion injury, malaria,mycobacterial infection, meningitis, psoriasis and other dermaldiseases, congestive heart failure, fibrotic disease, cachexia, graftrejection, cancer, tumor growth, undesirable angiogenesis, autoimmunedisease, opportunistic infections in AIDS, rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, other arthritic conditions,inflammatory bowel disease, Crohn's disease, ulcerative colitis,multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy,radiation damage, and hyperoxic alveolar injury. In addition, thecompounds can be used to treat other neurodegenerative diseases asexemplified by Alzheimer's disease, Parkinson's disease, head trauma,stroke and ALS.

The disclosed compounds can be used in combination with othercompositions and procedures for the treatment of diseases. For example,a tumor can be treated conventionally with surgery, radiation orchemotherapy in combination with an anti-angiogeniccompound/concentration and then, optionally the compound/concentrationcan be further administered to the subject to extend the dormancy ofmicrometastases and to stabilize and inhibit the growth of any residualprimary tumor. Alternatively, an angiogenic compound or angiogenicconcentration of a compound can be used in combination with otherangiogenesis stimulating agents. For example, thermal energy (in theform of resistive heating, laser energy or both) to create thermallytreated stimulation zones or pockets (optionally interconnected, atleast initially, by small channels) in the tissue for the introductionof blood born growth and healing factors, along with stimulatedcapillary growth surrounding the thermally treated zones. Suchstimulation zones allow increased blood flow to previously ischemicand/or nonfunctional tissue (such as cardiac tissue) with a concomitantincreased supply of oxygen and nutrients ultimately resulting in arevitalization of the treated sections the tissue when used incombination with the angiogenic compositions/concentrations. In otherembodiments, disclosed compounds exhibiting TNF-α inhibitory activitycan be combined with other TNF-α inhibitory agents, for example,steroids such as dexamethasone and prednisolone. When used for treatmentof a cancer, the compounds can be used in combination withchemotherapeutic agents and/or radiation and/or surgery.

Examples of other chemotherapeutic agents that can be used incombination with the disclosed compounds include alkylating agents,antimetabolites, natural products, kinase inhibitors, hormones and theirantagonists, and miscellaneous other agents. Examples of alkylatingagents include nitrogen mustards (such as mechlorethamine,cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkylsulfonates (such as busulfan), and nitrosoureas (such as carmustine,lomustine, semustine, streptozocin, or dacarbazine). Examples ofantimetabolites include folic acid analogs (such as methotrexate),pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs,such as mercaptopurine or thioguanine. Examples of natural productsinclude vinca alkaloids (such as vinblastine, vincristine, orvindesine), epipodophyllotoxins (such as etoposide or teniposide),antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin,plicamycin, or mitocycin C), and enzymes (such as L-asparaginase).Examples of kinase inhibitors include small molecule inhibitors (such asIressa, Tarceva, PKI-166, CI-1033, CGP-5923A, EKB-569, TAK165,GE-572016, CI-1033, SU5416, ZD4190, PTK787/ZK222584, CGP41251, CEP-5214,ZD6474, BIBF1000, VGA1102, SU6668, SU11248, CGP-57148, tricyclicquinoxalines, SU4984, SU5406, Gleevec, NSC680410, PD166326, PD1173952,CT53518, GTP14564, PKC412, PP1, PD116285, CGP77675, CGP76030, CEP-701,and CEP2583), ligand modulators (such as Bevacizumanb, MV833, SolubleFlt-1 and Flk-1, VEGF Trap, GFB 116, NM3, VEGF 121-diptheria toxinconjugate and Interferon-α), and monoclonal antibodies against receptors(such as Cetuximab, ABX-EGF, Y10, MDX-447, h-R3, EMD 72000, herceptin,MDX-H210, pertuiumab, IMC-1C11, and MF1). Examples of hormones andantagonists include adrenocorticosteroids (such as prednisone),progestins (such as hydroxyprogesterone caproate, medroxyprogesteroneacdtate, and magestrol acetate), estrogens (such as diethylstilbestroland ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens(such as testerone proprionate and fluoxymesterone). Examples ofmiscellaneous agents include platinum coordination complexes (such ascis-diamine-dichloroplatinum II, which is also known as cisplatin),substituted ureas (such as hydroxyurea), methyl hydrazine derivatives(such as procarbazine), vaccines (such as APC8024), AP22408,B43-genistein conjugate, paclitaxel, AG538, and adrenocroticalsuppressants (such as mitotane and aminoglutethimide). In addition, thedisclosed compounds can be combined with gene therapy approaches, suchas those targeting VEGF/VEGFR (including antisense oligonucleotidetherapy, Adenovirus-based Flt-1 gene therapy, Retrovirus-base Flk-1 genetherapy, Retrovirus-based VHL gene therapy, and angiozyme) and IGF-1R(including INX-4437). Examples of the most commonly used chemotherapydrugs that can be used in combination with the disclosed tricycliccompounds agent include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan,CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU,Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol, Velban, Vincristine,VP-16, Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11),Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin),Xeloda (Capecitabine), Zevelin and calcitriol.

The disclosed compounds also can be combined with radiotherapy employingradioisotopes (such as ³²P, ⁹⁰Y, ¹²⁵I, ¹³¹I and ¹⁷⁷Lu), particle beams(such as proton, neutron and electron beams) and electromagneticradiation (such as gamma rays, x-rays and photodynamic therapy usingphotosensitizers and visible or ultraviolet rays).

Additionally, the disclosed compounds can be combined withpharmaceutically acceptable excipients, and optionally sustained-releasematrices, such as biodegradable polymers, to form therapeuticcompositions. Therefore, also disclosed are pharmaceutical compositionsincluding one or more of any of the compounds disclosed above and apharmaceutically acceptable carrier. The composition may comprise a unitdosage form of the composition, and may further comprise instructionsfor administering the composition to a subject to inhibit angiogenesis,for example, instructions for administering the composition to achievean anti-tumor effect or to inhibit a pathological angiogenesis. Inparticular embodiments, the pharmaceutical composition may comprise oneor more of 1-Thioxo-3-oxo-2-(2-oxo-6-thioxopiperidin-3-yl)isoindoline,1,3-Dioxo-2-(2,6-dithioxopiperidin-3-yl)isoindoline,1-Thioxo-3-oxo-2-(2,6-dithioxopiperidin-3-yl)isoindoline,N-(2,6-dioxopiperidin-3-yl)-2,3-naphthalenedicarboxamide,1,3-Dioxo-2-(2,6-dioxopiperidin-3-yl)-5-azaisoindoline,1,3-Dioxo-2-(1-phenethyl-2,6-dioxopiperidin-3-ypisoindoline,2-Acetoxy-N-(2,6-dioxopiperidin-3-yl)benzamide,2-(2-Oxo-6-thioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione, Dimethyldihydro-1-oxo-3-thioxo-2H-isoindo1-2-yl)-pentanedioate, Dimethyl2-(1,3-dihydro-1,3-dithioxo-2H-isoindo1-2-yl)-pentanedioate,2-(1,3-Dihydro-1-oxo-3-thioxo-2H-isoindo1-2-yl)-pentanedioic acid,2,3-Dihydro-3-thioxo-2-(2,6-dioxo-3-piperidinyl)-1H-isoirtdol-1-one,2-(2, 6-Dithioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione,2,3-Dihydro-3-thioxo-2-(2-oxo-6-thioxo-3-piperidinyl)-1H-isoindol-1-one,2,3-Dihydro-3-thioxo-2-(2,6-dithioxo-3-piperidinyl)-1H-isoindol-1-one,2-(3-Cyclohexenyl)-1H-isoindol-1,3(2H)-dithione,2-(3-Cyclohexenyl)-1H-isoindole-1,3(2H)-dione,2-(3-Cyclohexenyl)-1H-isoindo1-1,3(2H)-dithione,2,3-Dihydro-3-thioxo-2-(3-cyclohexenyl)-1H-isoindol-1-one,3-(2,6-Dioxopiperidin-3-yl)benzoxazine-2,4-dione,1-(2,6-Dioxo-3-piperidinylidene)-3-oxoisoindoline,6-Thioxo-2-piperidinone, 2,6-Piperidinedithione, monothiophthalimide,dithiophthalimide, N-phenethylphthalimide,3-Benzylimino-2-benzyl-2,3-dihydroisoindol-1-one, 3-Camphanicamino-2,6-piperdinedione and3-[2′,6′-pipericlinedion-3′-yl]-7-amino-2H-1,3-benzoxazine-2,4(3H)-dione;and a pharmaceutically acceptable carrier. In more particularembodiments, the disclosed compositions are compounded for oraladministration, and such oral dosage forms can include one or more ofany of the disclosed compounds including those compounds particularlydisclosed by their IUPAC names above. Such pharmaceutical compositionsmay be used in methods for modulating angiogenesis or TNF-α activity ina subject by administering to the subject a therapeutically effectiveamount of the composition.

As is demonstrated in the Examples that follow, thionation ofthalidomide analogs to replace carbonyl groups with thiocarbonyl groupscan provide thalidomide analogs with increased TNF-α activity, increasedangiogenic activity or increased anti-angiogenic activity. Thus,although in certain structures the compounds are shown with carbonylgroups, it is to be understood that thionated derivatives of suchcompounds are also part of the disclosure.

4. EXAMPLES Example 1 Improved Synthesis of Thalidomide

With reference to Scheme 1 below, t-Butoxycarbamate 2, on reaction withcarbodiimide in THF, gave imide 3. Imide 3 was deprotected withtrifluoroacetic acid in CH₂Cl₂ at room to yield aminoglutarimidetrifluoroacetate 4. Without further purification, compound 4 was reactedwith phthalic anhydride in refluxing THF in the presence oftriethylamine to afford thalidomide 1 in the total yield of 24% from 2.This procedure is much more practical and efficient than several priorreported synthetic routes for the preparation of thalidomide.

2,6-Dioxo-3-(t-butoxycarbonylamino)piperidine (3) was prepared andisolated as follows. A solution of N-(t-butoxycarbonyl)-L-glutamine(4.92 g) and carbonyl diimidazole (1.70 g) in THF (100 mL) was refluxedfor 9 h. The solvent was removed and the crude product wasrecrystallized from hot EtOAc to give compound 3 (2.04 g, 45%) as whitecrystals: mp 214-215° C.; ¹H NMR (DMSO-d₆) δ 4.22 (dd, J=6.2 Hz, J=11.0Hz, 1H), 2.77-2.65 (m, 1H), 2.45 (m, 1 H), 1.96-1.87 (m, 2H), 1.40 (s,9H); MS (CI/CH₄) 227 [M-1]⁺.

2,6-Dioxo-3-aminopiperidine trifluoroacetate (4) was prepared andisolated as follows. Compound 3 (59 mg) was suspended in CH₂Cl₂ (5 mL).CF₃COOH (0.5 mL) was added. The reaction solution was stirred at roomfor 4 h. The solvent was removed to give 4 (62 mg, 99%): ¹H NMR(DMSO-d₆) δ 11.42 (s, 1H), 8.70 (br, 2H), 4.31 (dd, J=5.4 Hz, J=13 Hz),2.88-2.72 (m, 2H), 2.25-2.09 (m, 2H).

Thalidomide (1) was prepared and isolated as follows. A mixture of 4,phthalic anhydride and Et₃N in THF was refluxed for two days. Thereaction mixture was concentrated and purification by columnchromatography (eluent CH₂Cl₂/EtOAc=6:1) gave thalidomide (104 mg, 54%)as white crystals.

Example 2 Synthesis of Aromatic Thalidomide Analogs

With reference to Scheme 2 below, dimethylether 5 was obtained bycondensation of aminopyridine with phthalic anhydride in refluxing AcOHin the presence of sodium acetate. On standing with HBr in glacial AcOHsolution (30%) at room for 18 h, selective ether cleavage of 5 wasaccomplished to give compound 6. The structure of compound 6 wasdetermined by mass spectroscopy, 1D NMR and 2D NMR. The molecular ionfor compound 6 is 270 amu, demonstrating that only one methyl ether wascleaved. 2D NOESY showed that protons on the methoxy group correlatedwith H-5, indicating that the 2-methoxy was selectively cleaved and thatthe 6-methoxy remained. When the reaction elevated to 70° C., bothmethyl ethers were cleaved with HBr/HOAc solution (30%) to give diol 7.

1,3-Dioxo-2-(2,6-dimethoxypyridin-3-yl)-isoindoline (5) was prepared andisolated as follows. A mixture of phthalic anhydride (0.89 g, 6 mmol),3-amino-2,6-dimethoxypyridine monohydrochloride (95%, 1 g, 5 mmol) andsodium acetate (0.49 g, 6 mmol) in glacial acetic acid (50 ml) wasrefluxed for 3 h. The solvent was removed under vacuum. The residue wasdissolved in dichloromethane (200 ml) and washed with water (100 ml×3),dried over Na₂SO₄ and concentrated to give the crude product. The crudeproduct was recrystallized with ethyl acetate to give 5 (1.345 g, 90%)as a pale pink crystals: nip 182-183° C.; ¹H NMR (CDCl₃) δ 7.96-7.90 (m,2H), 7.80-7.76 (m, 2H), 7.44 (d, J=8.1 Hz, 1H), 6.42 (d, J=8.1 Hz, 1H,3.95 (s, 3 H), 3.91 (s, 3 H); ¹³C NMR (DMSO-d₆) δ 166.5, 160.6, 156.1,140.1, 132.8, 12914, 121.4, 104.6, 99.3, 51.7, 51.5; MS (CI/CH₄) 285[M+1]⁺. Anal. Calcd for C₁₅H₁₂N₂O₄: C, 63.38; H, 4.25; N, 9.85. Found:C, 63.57; H, 4.18; N, 9.65.

1,3-Dioxo-2-(2-hydroxy-6-methoxypyridin-3-yl)-isoindoline hydrobromide(6) was prepared and isolated as follows. To a flask were added2,6-dimethoxy-3-phthalimidopyridine (155 mg, 0.546 mmol) and hydrogenbromide solution in acetic acid (30%, 6 ml). The mixture was stirred atroom under N₂ for 18 h. Dry ether was added slowly until the solutionbecame cloudy. White crystals were precipitated, filtered and washedwith ether and ethyl acetate to afford 6 (127 mg, 67%) as white powderycrystals: mp 250° C.; ¹H NMR (DMSO-d₆) δ 7.97-7.94 (m, 211), 7.91-7.88(m, 211), 7.64 (d, J=8.2 Hz, 1H), 6.25 (d, J=8.2 Hz, 1H), 3.86 (s, 311);¹³C NMR (DMSO-d₆) δ 167.5, 162.1, 159.1 , 142.8, 135.4, 132.1, 123.7,108.2, 96.4, 54.8; MS (CI/CH₄) 270 [M]⁺.

1,3-Dioxo-2-(2,6-dihydroxypyridin-3-yl)-isoindoline hydrobromide (7) wasprepared and isolated as follows. To a flask were added2,6-dimethoxy-3-phthalimidopyridine (150 mg, 0.528 mmol) and hydrogenbromide solution in acetic acid (30%, 6 ml). The mixture was stirred atan 70° C. oil bath under N₂ for 54 h. The mixture was cooled to room ,dry ether was added, and the supernatant liquid was decanted. Then ethylacetate was added, solid precipitated, filtered and washed with ethylacetate to afford 7 (126 mg, 71%) as a white solid: ¹H NMR (CD₃OD) δ7.83-7.77 (n, 4H), 6.37 (d, J=8.1 Hz, 1H), 6.37 (d, J=8.1 Hz, 1H); MS(CI/CH₄) 256 [M]⁺; HRMS (DEI) m/z calcd for C₁₃H₈N₂O₄ 256.0484, found256.0483.

Example 3 Synthesis of N-Substituted Thalidomide Analogs

With reference to Scheme 3 below, a mixture of N-phthaloyl-DL-glutamicanhydride and phenethylamine was heated in a 177° C. oil bath. Thereaction mixture was purified by chromatography on a silica gel columnto afford N-phenethylthalidomide (8) and N-phenethylphthalimide (9).

1,3-Dioxo-2-(1-phenethyl-2,6-dioxopiperidin-3-yl)isoindoline (8) wasspecifically prepared and isolated as follows. A mixture ofN-phthaloyl-DL-glutamic anhydride (300 mg, 1.13 mmol) and phenethylamine(139 mg, 1.13 mmol) was stirred in a 177° C. oil bath for two hours. Thereaction mixture was cooled down and purified by column chromatography,first using petroleum ether/dichloromethane (1:5) as an eluent to affordN-phenethyl phthalimide as a pale yellow solid ¹H NMR (CDCl₃) δ7.78-7.77 (m, 2H), 7.65-7.62 (m 2 H), 7.22-7.16 (m, 5 H), 3.83 (t, 2H),2.92 (t, 2 H)), and then using dichloromethane as an eluent to affordN-phenethyl thalidomide as a syrup that was then recrystallized fromether to provide white crystals [(139 mg, 34%): mp 122-123° C.; ¹H NMR(CDCl₃) δ 7.84-7.81 (dd, J=3.1 Hz, J=5.4 Hz, 2H), 7.72-7.69 (dd, J=3.1Hz, J=5.4 Hz, 2 H), 7.20-7.14 (m, 5 H), 4.89 (dd, J=5.4 Hz, J=12.5 Hz, 1H), 4.01-3.92 (m, 2 H), 2.90-2.63 (m, 5H), 2.06-2.02 (m, 1 H); Anal.Calcd for C₂₁H₁₈N₂O₄: C, 69.60; H, 5.01; N, 7.73. Found: C, 69.40; H,5.13; N, 7.74].

Example 4 Synthesis of Azathalidomides

With reference to Scheme 4 below, azathalidomide was prepared fromaminoglutarimide and commercial pyridine-3,4-dicarboxylic anhydride.Cbz-aminoglutarimide was deprotected by hydrogenolysis with catalystpalladium hydroxide on carbon (10%) to form aminoglutarimide.Pyridine-3,4-dicarboxyic anhydride was refluxed with aminoglutarimide inthe presence of triethylamine to yield azathalidomide 11 in the totalyield of 17% from Cbz-aminoglutarimide.

1,3-Dioxo-2-(2,6-dioxopiperidin-3-yl)-5-azaisoindoline (11) was preparedspecifically as follows. A mixture of Cbz-aminoglutarimide (302 mg) andpalladium hydroxide on carbon (20%) in 2-propanol (20 ml) was stirredunder H₂ for one day. The reaction mixture was filtered through celiteand washed with 2-propanol and methanol. The combined filtrate wasconcentrated to afford crude 3-amino-1,6-dioxopiperidine as syrup. Tothe flask containing 3-amino-1,6-dioxopiperidine was added3,4-pyridinedicarboxylic anhydride (205 mg), triethylamine (0.16 ml) andTHF (10 ml). The mixture was refluxed for one and a half days. Thesolvent was removed under vacuum. The residue was purified by columnchromatography using CH2Cl₂:MeOH (10:1) as eluent to affordazathalidomide (52 mg) in the yield of 17% from Cbz-aminoglutarimide asa pale purple solid: mp 233-235° C., ¹HNMR (DMSO) δ 11.18 (s, 1H), 9.21(s, 1H), 9.17 (d, J=4.8 Hz, 1 H), 7.98 (d, J=4.8 Hz, 1 H), 5.23 (dd,J=5.4 Hz, J=12.8 Hz, 1 H), 2.96-2.85 (m, 2 H), 2.60-2.51 (m, 1 H),2.12-2.07 (m, 1H); MS (CI/CH₄) m/z 259 [M]⁺; Anal. Calcd for C₁₂H₉N₂O₄:C, 55.60; H, 3.50; N, 16.21. Found: C, 55.36; H, 3.44; N, 15.94.

Example 5 Synthesis of Acetoxythalidomide Analogs

With reference to scheme 5 above, acetoxythalidomide was prepared andisolated as follows. First, 3-Acetoxyphathalic anhydride was prepared byrefluxing a mixture of 3-hydroxyphthalic anhydride (150 mg), aceticanhydride (2 mL), and NaOAc (150 mg) for 8 h. The reaction mixture wasfiltered. The filtrate was concentrated and washed with dry ether togive a pale yellow solid (127 mg, 68%). ¹H NMR (DMSO) δ 8.25 (d, J=7.9Hz, 1H), 8.18 (dd, J=0.9 Hz, J=7.5 Hz, 1H), 7.97 (dd, J=0.9 Hz, J=7.9Hz, 1H), 2.59 (s, 3H).

1,3-Dioxo-2-(2,6-dioxopiperidin-3-yl)-4-acetoxyisoindoline was preparedand isolated as follows. A mixture of 3-acetoxyphthalic anhydride (40mg), aminoglutarimide trifluoroacetate (47 mg), and NaOAc (32 mg) inacetic acid (2 mL) was refluxed for 5 h. The solvent was evaporated,water (10 mL) was added, and the resulting solution was stirred forseveral minutes. The solid was filtered out and recrystallized fromethyl acetate to give1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-acetoxyisoindoline as paleyellow crystals (35 mg, 66%): ¹H NMR (DMSO) δ 11.16 (s, 1H), 11.07 (s,1H), 7.64 (t, J=7.2 Hz, 1H), 7.22-7.31 (m, 2H), 5.05 (dd, J=5.4 Hz,J=12.5 Hz, 1H), 2.87-2.92 (m, 2H), 2.48 (s, 3H), 2.08-2.00 (m, 2H).

Example 6 Synthesis of Benzothalidomides

With reference to Scheme 6 below, 1,8-Naphthalic anhydride on heatingwith amine 4 in the presence of triethylamine in THF gave 12.Naphthalene-2,3-dicarboxylic acid was converted to the anhydride 13which was reacted with aminoglutarimide trifluoroacetate 4 to affordbenzothalidomide 14. Spectral data, including mass spectra and NMR, aswell as combustion analyses were in accord with the structures assignedto these products.

Specifically, N-(2,6-dioxopiperidin-3-yl)-1,8-naphthatimide (12) wasprepared and isolated as follows. A mixture of amine 4 (0.877 mmol),1,8-naphthalic anhydride (174 mg, 0.879) and triethylamine (1.22 ml) inTHF (10 ml) was refluxed for 20 h. The solvent was removed and theresidue was suspended in acetic anhydride and refluxed for 20 minutes.Ethanol (5 ml) was added at 80° C. and stirred for 30 min. On coolingthe product was collected by filtration, and washed with EtOAc to givecompound 12 (227 mg, 84%) as a pale green solid: mp>300° C.; ¹H NMR(DMSO-d₆)δ 11.03 (s, 1H), 8.61-8.47 (m, 4H), 7.92 (dd, J=7.3 Hz, J=13.5Hz, 2H), 5.85 (dd, J=5.4 Hz, J=11.3 Hz, HD, 3.01-2.88 (m, 1H), 2.73-2.61(m, 2H), 2.08-1.99 (m, 1H). MS (DEI) m/z 309 [M+1]⁺; HRMS (DEI) m/zcalcd for C₁₇H₁₃N₂O₄ 309.0875, found 309.0874; Anal. Calcd forC₁₇H₁₂N₂O₄: C, 66.23; H, 3.92; N, 9.09. Found: C, 65.97; H, 3.99; N,8.91.

N-(2,6-dioxopiperidin-3-yl)-2,3-naphthalenedicarboxamide (14) wasprepared and isolated as follows. A mixture of2,3-naphthalenedicarboxylic acid (199 mg, o.875 mmol) and aceticanhydride (2 mL) was refluxed for 30 min. The reaction mixture wascooled down, and the solid was collected by filter to afford anhydride13 (0.133 g, 77%) as a white solid. To a solution of aminoglutarimidetrifluoroacetate (163 mg) and triethylamine (1 mL) in THF (10 mL) wasadded anhydride 13 (133 mg). The mixture was refluxed for 16 h. Thesolvent was removed under vacuum, and the residue was dissolved inEtOAc, washed with saturated aqueous NaHCO₃ solution and H₂O, dried andconcentrated. The residue was purified by flash chromatography to givecompound 14 as a white solid (146 mg, 70%). mp>300° C.; ¹H NMR(DMSO-d₆)δ 11.3 (s, 1H), 8.60 (s, 2H), 8.30 (dd, J=3.3 Hz, J=6.1 Hz,2H), 7.82 (dd, J=3.2 Hz, J=6.2 Hz, 2H), 5.24 (dd, J=5.6 Hz, J=13.0 Hz,1H), 2.99-2.86 (m, 2H), 2.66-2.57 (m, 2H), 2.12-1.99 (m, 1H). MS (DEI)m/z 308 [M]⁺; FIRMS (DEI) m/z calcd for C₁₇H₁₂N₂O₄ 308.0797, found308.0798; Anal. Calcd for C₁₇H₁₂N₂O₄-0.25H₂O: C, 65.28; H, 4.03; N, 8.96Found: C, 65.42; H, 3.93; N, 8.94.

Example 7 Synthesis of Sulfur Analogs of Thalidomide

With reference to Scheme 7 below, reaction of thalidomide 1 withLawesson's reagent, when stirred in benzene at 80° C. for 48 h, yieldedthionamide 15 in a yield of 38%. In addition to monothiothalidomide, atrace of dithionimide 16 (1.6%) was also obtained. However, for thepreparation of dithionimide, the yield proved to be very low (less than2%) when the reaction of monothiothalidomide with Lawesson's reagent wasperformed between 80° C. to 120° C. The situation changed greatly whenorganic base was added to the reaction mixture. Thus, thionation ofmonothiothalidomide 15 with Lawesson's reagent in toluene was carriedout at 110° C. in the presence of pyridine to give dithionimide 16 (45%)and dithionimide 17 (31%). The structures of these sulfur-substitutedthalidomides were identified by mass spectra, 1DNMR and 2DNMR.Thalidomide was heated with Lawesson's reagent at 110° C. in thepresence of morpholine to afford dithionimide 16 and trithionimide 18.

1,3-Dioxo-2-(2-oxo-6-thioxopiperidin-3-yl)isoindoline (15) wassynthesized and isolated as follows. A mixture of thalidomide (170 mg,0.658 mmol) and Lawesson's reagent (293 mg, 0.724 mmol) in benzene (50ml) was stirred in a 80° C. oil bath for 2 days. The solvent was removedunder vacuum. The residue was purified by column chromatography usingCH₂CH₂/petroleum ether (5:1) as eluent to afford compound 16 (3 mg,1.6%) as a red solid and then, using CH₂Cl₂ as eluent, to affordcompound 15 (68 mg, 38%) as a yellow solid: mp 225-226° C.; ¹H NMR(DMSO-d₆)δ 12.83 (s, 1H), 8.00-7.92 (m, 4H), 5.32 (dd, J=5.6 Hz, J=12.9Hz, 1H), 3.28-3.25 (m, 1H), 2.60-2.54 (m, 2H), 2.17-2.10 (m, 1H); ¹³CNMR (DMSO-d₆) δ 208.7(C-6′), 165.3(C-2′), 165.2(C-1 & C-3), 133.1(C-5 &C-6), 129.3 (C-3a, Ta), 121.7 (C-4 & C-7), 46.9 (C-3′), 38.9 (C-5′),21.79 (C-4′); MS (CI/CH₄) m/z 274 [M]⁺; Anal. Calcd for C₁₃H₁₀N₂O₃S: C,56.92; H, 3.67; N, 10.21 Found: C, 56.89; H, 3.78; N, 10.15.

1-Thioxo-3-oxo-2-(2-oxo-6-thioxopiperidin-3-yl)isoindoline (16) and1,3-dioxo-2-(2,6-dithioxopiperidin-3-yl)isoindoline (17) weresynthesized as follows. A mixture of 15 (146 mg, 0.533 mmol), Lawesson'sreagent (108 mg, 0.267 mmol) and pyridine (21 μl) in toluene was stirredat 110° C. limier an atmosphere of N₂ for 12 h. Thereafter, moreLawesson's Reagent (108 mg, 0.267 mmol) and pyridine (21 μl) were added.The reaction mixture was stirred for a further 12 h. The solvent wasremoved under vacuum and the residue was purified by columnchromatography (eluent CH₂Cl₂/petroleum ether=2:1, 10:1, thenCH₂Cl₂/EtOAc=10:1) to afford 16 (30 mg, 45%) and 17 (21 mg, 31.5%).Starting material 15 (83 mg) was also recovered.

Compound 16: (yellow solid): mp 263-265° C.; ¹H NMR (CDCl₃) δ 7.78-7.74(m, 2 H), 7.66-7.63 (m, 2 H), 5.00 (dd, J=4.9 Hz, 11.9 Hz, 1 H),3.43-3.35 (m, 1 H), 2.95-2.84 (m, 2 H), 2.08-2.06 (m, 1 H); MS (DEI) m/z290 [M]⁺; HRMS (DEI) m/z calcd for C₁₃H₁₀N₂O₂S₂ 290.0184, found290.0185; Anal. Calcd for C₁₃H₁₀N₂O₂S₂: C, 53.77; H, 3.47; N, 9.65Found: C, 53.38; H, 3.29; N, 9.50.

Compound 17: (red solid): mp 240-242° C.; ¹H NMR (CDCl₃) δ 9.44 (s, 1H), 8.05-8.02 (m, 1 H), 7.86-7.76 (m, 3 H), 5.75-5.64 (m, 1 H),3.57-3.52 (m, 1 H), 3.09-2.99 (m, 2 H), 2.19- 2.12 (m, 1 H). ¹³C NMR(DMSO): 208.16, 207.98, 166.10, 165.39, 134.32, 133.11, 132.42, 124.30,122.15, 121.11, 49.64, 21.29; MS(DEI) m/z 291 [M]⁺; HRMS (DEI) m/z calcdfor C₁₃H₁₁N₂O₂S₂ 291.0262, found 291.0264; Anal. Calcd forC₁₃H₁₀N₂O₂S₂.0.5H₂O: C, 52.15; H, 3.70; N, 9.36 Found: C, 52.25; H,3.44; N, 9.07.

1-Thioxo-3-oxo-2-(2,6-dithioxopiperidin-3-yl)isoindoline (18) wasprepared and isolated as follows. A mixture of thalidomide (100 mg),Lawesson's reagent (157 mg) and morpholine (35 μl) in toluene (10 mL)was stirred at 105° C. under the atmosphere of N₂ for 24 h. The solventwas removed under vacuum and the residue was purified by columnchromatography, using CH₂Cl₂:petroleum ether (1:1) as eluent, to affordcompound 18 (13 mg, 11%) as red crystals: mp 244° C.; ¹H NMR (CDCl₃) δ10.81 (s, 1H), 8.05-8.01 (m, 1H), 7.91-7.75 (m, 3H), 5.92 (m, 1H),3.57-3.52 (m, 1H), 3.13-2.97 (m, 2H), 2.18-2.15 (m, 1H); MS(DEI) m/z 306[M]⁺; HRMS (DEI) m/z calcd for C₁₃H₁₀N₂OS₃ 305.9955, found 305.9951;Anal. Calcd for C₁₃H₁₀N₂OS₂.0.5H₂O: C, 49.49; H, 3.51; N, 8.88 Found: C,49.85; H, 3.24; N, 8.88. Then, CH₂Cl₂ was used as eluent to providecompound 16 (31 mg, 28%) as yellow crystals.

Example 8 Synthesis of Benzoxazine-2,4-diones

With reference to Scheme 8 below, salicylic acid was treated with ethylchloroformate, and then this reaction mixture was evaporated at reducedpressure to remove any unreacted ethyl chloroformate. Stirring theresulting residue with amine in the presence of triethylamine affordedsubstituted benzoxazine-2,4-diones

3-(2,6-Dioxopiperidin-3-yl)benzoxazine-2,4-dione (20) was prepared andisolated as follows. To a cold ice/salt solution of salicylic acid (100mg) and triethylamine (303 ml) in chloroform (10 mL) was added ethylchloroformate (157 ml). The reaction mixture was allowed to warm to roomtemperature, and, thereafter, stirring was continued for 3 h. Thesolvent was removed under vacuum to give crude 19. Without furtherpurification, crude compound 19 was dissolved in CHCl₃ and cooled withice. To the ice cold solution was added amine (95 mg). The reactionmixture was allowed to warm to ambient and stirred at room overnight.The white solid precipitated, collected by filtration and washed withchloroform to give compound 20 (79 mg, 74%) as a white crystals: mp 264°C.; ¹H NMR (DMSO-d₆)δ 11.18 (s, 1H), 8.07-7.85 (m, 2H), 7.50 (d, J=8.5Hz), 5.78-5.75 (m, 0.6H), 5.49-5.47 (m, 0.4H), 2.90-2.87 (m, 1H), 2.05(m, 1H); ¹³C NMR (DMSO-d₆)δ 173.0 (0.6C), 172.9 (0.4C), 169.9 (0.6C),169.6 (0.4C), 160.8 (0.6C), 159.8 (0.4C), 152.5 (1C), 148.4 (0.4C),146.5 (0.6C), 137.2 (1C), 128.1 (0.6C), 127.6 (0.4C), 126.1 (1C), 116.8(1C), 114.5 (0.4C), 113.9 (0.6C), 54.1 (0.4C), 51.4 (0.6C), 31.0 (1C),21.2 (1C). MS(DEI) m/z 274 [M]⁺; HRMS (DEI) m/z calcd for C₁₃H₁₀N₂O₅274.0590, found 274.0582; Anal. Calcd for C₁₃H₁₀N₂O₅: C, 56.94; H, 3.68;N, 10.22 Found: C, 56.51; H, 3.77; N, 9.95.

Example 9 Synthesis of 1-(2,6-Dioxo-3-piperidinylidene)-3-oxoisoindoline

With reference to Scheme 9 below, monothiophthalimide (21) was stirredwith 3-bromoglutarimide (22) in the presence of Na₂CO₃ in an Eschenmosercoupling reaction. Thus, compound 23 was formed by alkylation ofmonothiophthalimide with 3-bromoglutarimide, followed by elimination ofsulfur.

1-(2,6-Dioxo-3-piperidinylidene)-3-oxoisoindoline (23) was specificallyprepared and isolated as follows. A mixture of 21 (16 mg, 0.1 mmol), 22(19 mg, 0.1 mmol), and potassium carbonate (100 mg) in anhydrous THF wasrefluxed for 7 h. Thin-layer chromatography (TLC) showed that thestarting materials had disappeared. Ethyl acetate (20 ml) and water (10ml) were added. The organic layer was separated, dried over Na₂SO₄ andconcentrated under vacuum. The residue was purified by chromatographyusing petroleum ether/ethyl acetate (first 2:1 then 1:2) to give 23 (14mg, 58%) as yellow crystals: mp 295° C.; ¹HNMR (DMSO-d₆): 11.05 (s, 1H), 10.29 (s, 1 H), 8.13 (d, J=7.6 Hz, 1 H), 7.89 (d, J=7.2 Hz, 1 H),7.80 (m, 1 H), 7.73 (n, 1 H), 3.20 (t, J=7.0 Hz, 2 H), 2.67 (t, J=7.0Hz, 2 H). ¹³C NMR (DMSO-d₆): 172.6, 169.0, 167.3, 142.7, 136.1, 134.3,131.7, 130.1, 126.4, 124.1, 104.6, 21.2, 11.7. MS(DEI) m/z 242 [M]⁺;HRMS (DEI) m/z calcd for C₁₃H₁₀N₂O₃ 242.0691, found 242.0687.

Example 10 Salicylamide Analogs

Reaction of commercial acetylsalicyloyl with aminoglutarimidetrifluoroacetate was carried out to give acetylsalicylamide 24 accordingto Scheme 10 below.

More specifically, 2-acetoxy-N-(2,6-dioxopiperidin-3-yl)benzamide (24)was prepared as follows. To an ice cold solution ofacetylsalicyloylchloride (252 mg) and triethylamine (0.58 mL) inchloroform (30 mL) was added 3-aminoglutaride trifluoroacetate (207 mg).The reaction was allowed to warm to room and stirring was continuedovernight. The solvent was removed and recrystallization from ethylacetate gave compound 24 as white crystals (0.36 g, 98%): ¹H NMR(DMSO-d₆)δ 11.00 (s, 1H), 8.73 (d, J=8.3 Hz, 1H), 7.81 (dd, J=1.6 Hz,J=7.7 Hz, 1H), 7.72 (m, 1H), 7.54 (m, 1H), 7.38 (dd, J=0.9 Hz, J=8.1 Hz,1H), 4.95-4.82 (m, 1H), 2.96-2.90 (m, 1H), 2.43 (s, 3H), 2.18-2.15 (m,2H).

Example 11 Synthesis of Thiothalidomides and Determination of TheirTNF-α Inhibitory Activity

A series of thiothalidomides and analogs were designed to explore theiraction on inhibition of TNF-α. Monothiothalidomide 205 (same as compound15 in Example 7) was prepared as shown in Scheme 11.tert-Butoxycarbonyl-L-glutamine 202 was refluxed with carbonyldiimidazole (CM) in THF, and cyclized to afford imide 203 (Muller etal., “Amino-substituted thalidomide analogs: potent inhibitors of TNF-αproduction,” Bioorg. Med. Chem, Lett. 9, 1625-1630, 1999).

Imide 203 then was treated with trifluoroacetic acid in CH₂Cl₂ to removethe protective group to generate aminoglutarimide trifluoroacetate 204.Without further purification, compound 204 was reacted with phthalicanhydride in refluxing THF in the presence of triethylamine to producethalidomide 201 (same as compound 1 in Example 7) in the total yield of31% from compound 202. Thalidomide 201 was thionated with Lawesson'sreagent (LR, Cava et al., “Thionation reaction of Lawesson's Reagents,”Tetrahedron, 41, 5061-5087, 1985, the entirety of which is incorporatedherein by reference) to generate a single new product that had astructure identified as 6′-thiothalidomide 205 by mass spectrometry and1D & 2D nuclear magnetic resonance spectroscopy. The position of thethiocarbonyl group was established from the heteronuclear multiple bondcorrelation (HMBC) cross peak of H-5′/C-6′.

The synthesis of 3-thiothalidomide 212 is shown in Scheme 12 below.N-Phthaloyl-L-glutamic acid 206 was esterified to afford diester 207.Compound 207 was thionated with LR at 110° C. to give compound 208 as amajor product. Concurrently, compound 209 was separated as a minorproduct by chromatography.

3-thiothalidomide, 212, could not be prepared through the cyclization ofcompound 208 with ammonia or amine as ammonia reacts with the thioamide;reaction of compound 208 with benzylamine produced the unexpectedcompound 210. In an alternative approach, compound 208 was hydrolyzedunder acidic conditions to give diacid 211. Compound 211 was thenreacted with trifluoroacetamide to generate 3-thiothalidomide 212 in thepresence of 1-hydroxybenzotriazole (HOBt) and1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI,Flaih et al., “An expeditious synthesis of cyclic imides,” TetrahedronLeft. 40, 3697-3698, 1999).

In the synthesis of dithiothalidomide, one method involved the reactionof monothiothalidomide with LR at reflux in toluene. Under suchconditions, 2′,6′-dithiothalidomide was obtained in a yield of less than2% (Scheme 13a). The yield was so low that improvement was desirable,and was undertaken by modifying the reaction conditions. It is believedthat the mechanism underlying the reaction between LR and a carbonylmoiety is that a highly reactive dithiophosphine ylide 214, rather thanLR itself, likely is the active thionating agent (Scheme 4, Cava et al.,“Thionation reaction of Lawesson's Reagents,” Tetrahedron, 41,5061-5087, 1985, the entirety of which is incorporated herein byreference). The Lewis base may be able to increase the reactivity of LRas the base may drive the unfavorable equilibrium and elevate theconcentration of the ylide 214. When pyridine was used as a catalyst forthionation, monothiothalidomide 205 was thionated with LR to produce twodithiothalidomides, 213 (same as compound 16in Example 7) and 215 (sameas compound 17 in Example 7), in yields of 45% and 31%, respectively(Scheme 13 b,c). Dithiothalidomide 213 was further thionated with LR inthe presence of the stronger base, morpholine, to givetrithiothalidomide 216 (same as compound 18 in Example 7) in a yield of65%.

Glutarimide 217 was thionated with LR in THF at room to afford compound218 as a major product. Glutarimide 217 also was refluxed with LR intoluene to produce dithioglutarimide 219 (Scheme 15). Reaction ofpotassium phthalimide with 3-bromocyclohexene in a Gabriel reaction gavecompound 221. Thereafter, thionation of compound 221 with LR affordedcompounds 222 and 223 (Scheme 16). Compounds 224 and 225 were preparedin a similar procedure to that used in the preparation of compounds 222and 223.

The structures of the thiothalidomide compounds of this Example aresummarized below.

The action of the these thiothalidomide analogs in inhibiting TNF-αsecretion was assessed in human peripheral blood mononuclear cells(PBMC) and the results are shown in Table 1. Freshly prepared PBMCs wereutilized in all studies. Blood, 40 ml, was drawn from a volunteer,immediately mixed with 50 U/ml Na heparin and was diluted to 50 ml totalvolume with sterile PBS. Samples, 20 ml, of this preparation then werelayered on 20 ml Ficoll-Paque and were centrifuged (800 g, 20 min) TheFicoll/serum interface, containing PBMCs, was collected, diluted to 200ml with PBS, and then was centrifuged (800 g, 15 min) to pellet thecells. Thereafter, the recovered pellet was re-suspended in 37° C.tissue culture medium (RPMI/1 mM Sodium pyruvate/10% heat inactivatedFBS/2 mM Glutamax) and placed on ice. Recovered cells were counted,pipetted (200 ul of 5×10⁵/ml) into 96 well plates, and incubated for anhour (37° C., 5% CO₂). Thereafter, appropriate concentrations of testcompounds or vehicle (10 ul DMSO) were added to duplicate wells.Following a further hour of incubation, a 10 ul sample oflipopolysaccharide (LPS)(100 ng/ml in supplemented medium) or vehiclewas added to induce stimulated and unstimulated cells, respectively, andthe cells were incubated overnight. Sixteen hours later, supernatantswere collected for quantification of TNF-α levels by ELISA assay(Pierce-Endogen human TNF-α mini kit, Rockford, Ill.) and the use ofspecific capture and detection monoclonal antibodies, M303E and M302B(Pierce-Endogen), respectively. ELISA plates were read at λ=450 nm andTNF-α levels were determined from a six-point calibration curve that wasrun concurrently with the test samples. The effect of test drugconcentrations on the cellular viability of PBMCs was assessed by MTSassay (Promega, Madison, Wis.) of the cells that provided thesupernatant samples assayed for TNF-α levels, described above. It shouldbe understood that this method can be used to test any of the disclosedcompounds as a screening assay for readily determining their TNF-αmodulating activity, and for selecting them for use in the disclosedmethod of treating a subject.

TABLE 1 Inhibition of LPS-induced TNF-α production in PBMC and cellviability % Inhibition IC₅₀ Cell viability Compound at 30 μM (μM) at 30μM at 3 μM at 0.3 μM 205 31 >30 >100 90 96 208 56 20 93 99 96 209 85 1057 86 89 211 20 >30 86 93 93 212 23 >30 94 100 94 213 52 20 69 87 94 21561 11 >100 87 94 216 79 6 94 86 90 218 15 >30 >100 84 86 219 75 8 >10098 99 222 86 15 50 94 96 223 85 16 57 89 99 224 95 3 54 83 83 22534 >30 >100 94 94

Thalidomide, 201, entirely lacked activity at 30 μM. A concentration of100 μM was required for significant activity (IC₅₀˜200 μM). Themonothiothalidomides, 6′-thiothalidomide 205 and 3-thiothalidomide 212showed only marginal activity at 30 μM with 31% and 23% inhibition ofTNF-α secretion, respectively. In contrast, the dithiothalidomides,including 2′, 6′-dithiothalidomide 213 and 3, 6′-dithiothalidomide 215,exhibited more potent inhibitory activities with IC₅₀ values of 20 μMand 11 μM, respectively. However, assessment of cell viability by MTSassay showed that 213 induced increasing cytotoxicity at higherconcentrations. Trithiothalidomide 216 inhibited TNF-α production withan IC₅₀ of 6 μM, without accompanying toxicity. Compared withthalidomide, 201, with an IC₅₀ of ˜200 μM for the inhibition of TNF-αsynthesis, trithiothalidomide 216 is over 30-fold more active. Hence,successive replacement of a carbonyl with a thiocarbonyl group led toimproved inhibitory activity compared to 201, unassociated withtoxicity. In this regard, the synthesized thiothalidomides possessedTNF-α lowering potency in the following decreasing order:trithiothalidomide 216>dithiothalidomide 215 and213>monothiothalidomides 205 and 212>thalidomide, 201.

A comparison of the physical properties of thalidomide, 201, andthiothalidomides shows that they have similar Van der Waals radii andbond angles, although the C═S bond is slightly longer than the C═O bond.Although not wishing to be bound by any particular theory, a possibleexplanation accounting for the elevated potency of the thiothalidomidesis that their enhanced lipophilicity and loss of hydrogen bond acceptorcapability potentially allows the attainment of higher intracellulardrug levels. Interestingly, compounds 208, 209 and 211 are thio analogsof hydrolysis metabolites of thalidomide. Assessment of their TNF-αinhibitory action determined that the monothio analog, 208, has an IC₅₀of 20 μM without toxicity; demethylation (211) lowered potency. Thedithio analog, 209, proved 2-fold more potent still than 208, butinduced cellular toxicity at lower concentrations. Intriguingly, thioanalogs 222 and 223, with a simplified glutarimide ring, were found tobe active TNF-α inhibitors, albeit with some toxicity at 30 μM, withIC₅₀ values (15 μM and 16 μM respectively) that were greater than 212(>30 μM) possessing a normal glutarimide ring.

In this regard, thalidomide is composed of two distinct moieties: theglutarimide and phthalimide rings. Thioglutarimides and thiophthalimideswere thus synthesized and evaluated to assess the effect of thio-analogsof these two moieties on TNF-α levels. Monothioglutarimide 218 minimallyinhibited TNF-α secretion at a concentration of 30 μM, howeverdithioglutarimide 219 exerted a potent inhibitory effect with an IC₅₀ of8 μM and a lack of toxicity. Surprisingly, such a simple structure,dithioglutarimide 219, proved to be 25-fold more active than thalidomide201. In contrast, 2′, 6′-dithiothalidomide 213, a phthalimidosubstituted dithioglutarimide, is less active than dithioglutarimide219, and induces toxicity at high concentration. Monothiophthalimide 225showed marginal TNF-α activity at a concentration of 30 μM withouttoxicity. Interestingly, however, dithiophthalimide 224 was found topossess potent activity with an IC₅₀ of 3 μM. Although it was associatedwith toxicity at 30 μM, its inhibition of TNF-α occurred at an order ofmagnitude lower concentration that was well tolerated.

As described, compounds 215, 216 and 219 potently inhibited TNF-αsecretion without toxicity. As a consequence, additional studies wereundertaken to elucidate the mechanism underpinning this action. Gene andprotein expressions are controlled at the level of transcription,post-transcription, RNA stability, and translation under differentphysiological stimuli. Recently, post-transcriptional pathways have beenrecognized to provide a major means of regulating eukaryotic geneexpression. In this regard, TNF-α and other cytokines and protooncogenesare known to be regulated at the post-transcriptional level. Multipleproteins, including the four cloned proteins AUF1, HuR, TTP and HuD havebeen shown to bind to a region of the mRNA that containsadenylate/uridylate (AU)-rich elements (AREs) in the 3′-untranslatedregion (UTR). These proteins mediate RNA turnover and decay, and hencetranslational efficiency. The stability of TNF-α mRNA is largelyregulated at its 3′-UTR, which contains a well characterized ARE.Although AREs are found in a number of different cytokine andprotooncogene RNAs, the pathways by which they induce degradation arehighly specific for a given ARE indicating some cellular specificity.When the AREs from different cytokines are complexed with AUF1,different binding affinities are observed. Notably, however, the highestaffinity for AUF1 is to human and then mouse TNF-α.

To determine the involvement of the 3′-UTR in the action of thethalidomide analogs, their ability to inhibit reporter gene activity incells containing the TNF-α 3′-UTR versus a control vector was assessed.The results are shown in FIG. 1. This cell-based assay utilized twostably transfected cell lines derived from the mouse macrophage line,RAW264.7. One line, designated “luciferase only” expressed a luciferasereporter construct without any UTR sequences. The other line, designated“luciferase +TNF-α UTR” expressed a luciferase reporter construct withthe entire 3′-UTR of human TNF-α inserted directly downstream of theluciferase coding region. Compounds were added in aconcentration-dependent manner, and at the end of the incubation period(16 h, 37° C., 5% CO₂) the media was removed, cells were lysed andluciferase activity was assayed with Steady-glo luciferase assay reagent(Promega) according to the supplier's directions. Background wassubtracted and data from this assay was expressed as a ratio of the+3′-UTR to −3′-UTR (control) values, and was expressed as a percent asshown in FIG. 1. In this manner, compounds that show a differentialeffect on the two cell lines, with and without a 3′-UTR, arehighlighted. The action of compounds 215, 216 and 219 in cells (mousemacrophage cell line, RAW264.7) possessing a luciferase reporter elementplus the of human TNF-α compared to cells lacking the 3′-UTR are shownin FIG. 1. Compounds 215, 216 and 219 exerted differential effect on thetwo cell lines in a dose-dependent manner, consistent with their abilityto inhibit TNF-α production via the 3′-UTR. All agents loweredluciferase reporter activity in cells stably expressing the 3′-UTR.Thalidomide lacked activity at 50 μM.

As TNF-α protein levels changed without significant alterations in mRNAlevels (data not shown), protein expression is presumably regulated viatranslational control (at the post-transcriptional level). There isprecedence for translational (protein) control through either the 3′- or5′-UTR regions of a number of critical proteins that are current drugtargets. For example, levels of the beta-amyloid precursor protein (APP)that is central to the development of AD can be regulated by either UTR.Turnover and translation of APP mRNA is regulated by a 29-nucleotideinstability element within the 3′-UTR, located 200 nucleotidesdownstream from the stop codon. This 3′-UTR element acts as an mRNAdestabilizer whose function can be inhibited by the presence of growthfactors. In contrast, different cytokines, including TNF-α, and iron canup regulate APP protein synthesis at the level of its 5′-UTR; where,interestingly, the anticholinesterase, phenserine, that is currently inclinical trials for AD, lowers APP protein levels with concurrentmaintenance of mRNA steady-state levels through translationalmodification within the same 5′-UTR element. A further example is thatof the human immunodeficiency virus 1 (HIV-1) Trans-activatingtransduction (tat) protein, which binds trans-activation-responsiveregion (TAR) RNA. Tat is brought into contact with the transcriptionmachinery after binding the TAR element, which is a 59-residue stem-loopRNA found at the 5′ end of all HIV-1 transcripts. Finally, thalidomide(201) has been reported to lower cyclooxygenase-2 (Cox-2) biosynthesisvia its 3′-UTR that appears to likewise contain an ARE that can regulateCox-2 mRNA stability. The studies of analogs 215, 216 and 219 confirmregulation of TNF-α protein levels by thalidomide (201) via its 3′-UTR,but whether or not the 5′-UTR contains a similar element that isaccessible to pharmacological manipulation remains to be determined, asdoes action against Cox-2.

In summary, disclosed thiothalidomide analogs include analogs that aremore potent inhibitors of TNF-α production in LPS-induced human PBMCsthan thalidomide 201. The isosteric replacement of successive carbonylgroups by a thiocarbonyl leads to an increasing inhibition with thenumber of moieties replaced (trithiothalidonaide 216>dithiothalidomide215 and 213>monothiothalidomides 205 and 212>thalidomide 201).

TNF-α has been validated as a drug target for two drugs on the market;Remicade (Cetocor, Malvern, Pa.; Schering-Plough, Orange, N.J.) andEnbrel (Amgen, Thousand Oaks, Calif.; Wyeth-Ayerst, Princeton, N.J.).However, both of these drugs are large macromolecules and hence requireinjection. In contrast, the small molecule drugs disclosed herein offera means to potently and safely inhibit TNF-α without injection, forexample, by oral administration.

SYNTHESIS AND CHARACTERIZATION DETAILS

General. Melting points were determined with a Fisher-Johns apparatusand are uncorrected. ¹H NMR, ¹³C NMR and 2D NMR were recorded on aBruker AC-300 spectrometer. Mass spectra and high resolution massspectra (HRMS) were recorded on a VG 7070 mass spectrometer and aAgilent Technologies 5973N GC-MS (CI). All exact mass measurements showan error of less than 5 ppm. Elemental analyses were performed byAtlantic Microlab, Inc., Norcross, Ga.

3-(tert-Butoxycarbonylamino)-2,6-piperidinedione (203). A mixture ofN-(tert-butoxy carbonyl)-L-glutamine (4.92 g, 20 mmol) and carbonyldiimidazole (3.24 g, 20 mmol) in THF (100 mL) was refluxed for 16 h.Thereafter, solvent was removed and the crude product was recrystallizedfrom hot EtOAc to give compound 203 (2.04 g, 45%) as white crystals: mp214-215° C.; ¹H NMR (DMSO-d₆)δ 4.22 (dd, J=6.2 Hz, J=11.0 Hz, 1H),2.77-2.65 (m,1H), 2,45 (m, 1 H), 1.96-1.87 (m, 2H), 1.40 (s, 9H); MS(CI/CH₄) m/z 227 [M-1]⁺.

2-(2-Oxo-6-thioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione (205).Compound 203 (1.14 g, 5 mmol) was suspended in CH₂Cl₂ (100 mL). To themixture was added CF₃COOH (10 mL) and this then was stirred at room for4 h. The solvent was evaporated to give crude 204 (1.25 g): ¹H NMR(DMSO-d₆)δ 11.42 (s, 1H), 8.70 (br, 2H), 4.31 (dd, J=5.4 Hz, J=13 Hz),2.88-2.72 (m, 2H), 2.25-2.09 (m, 2H). A mixture of crude 204 (1.25 g)and phthalic anhydride (0.89 g, 6 mmol) and Et₃N (1.39 ml, 10 mmol) inTHF (150 mL) was refluxed for two days. The reaction mixture wasconcentrated and the residue was crystallized from ethyl acetate to givethalidomide (201) (0.89 g, 69%) as white crystals; mp 276° C.(lit.276-279° C.). A mixture of thalidomide 201 (258 mg, 1 mmol) andLawesson's reagent (222 mg, 0.55 mmol) in toluene (50 ml) was stirred atreflux for 12 h; thereafter, solvent was removed under vacuum. Theresulting residue was purified by column chromatography using CH₂Cl₂ asthe eluent to afford compound 205 (200 mg, 73%) as a yellow solid: mp225-226° C.; ¹H NMR (DMSO-d₆)δ 12.83 (s, 1H, NH), 8.00-7.92 (m, 4H, Ph),5.32 (dd, J=5.6 Hz, J=12.9 Hz, 1H, H-3′), 3.28-3.25 (m, 2H, H-5′),2.60-2.54 (m, 1H, H-4′), 2.17-2.10 (m, 1H, H-4′); ¹³C NMR (DMSO-d₆)δ208.7(C-6′), 165.3(C-2′), 165.2(C-1 & C-3), 133.1(C-5 & C-6), 129.3(C-3a, C-7a), 121.7 (C-4 & C-7), 46.9 (C-3′), 38.9 (C-5′), 21.79 (C-4′);MS (CI/CH₄) m/z 274 (M⁺); Anal. (C₁₃H₁₀N₂O₃S) C, H, N.

Dimethyl 2-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)-pentanedioate (207).To a solution of N-phthaloyl-L-glutamic acid (200 mg, 0.72 mmol) inmethanol (10 mL) was added, dropwise, thionyl chloride (1 mL). Thereaction mixture was refluxed for 6 h. The solvent was removed underreduced pressure, dissolved in ethyl acetate (100 mL), and then washedwith saturated aqueous Na₂CO₃ solution (2×30 mL) and water (2×30 mL).The ethyl acetate layer was dried over Na₂SO₄ and then evaporated,leaving an oil, which upon purification by silica gel chromatography,using CH₂Cl₂:EtOAc (1:1) as the eluent, gave compound 7 (161 mg, 73%) asan oil; ¹H NMR (CDCl₃)δ 7.87-7.84 (m, 2H), 7.75-7.72 (m 2H), 4.91 (dd,J=5 Hz, J=9 Hz, 1H), 3.73 (s, 3H), 3.62 (s, 3H), 2.67-2.56 (m, 1H),2.51-2.44 (m, 1H), 2.41-2.35 (m, 2H).

Dimethyl 2-(1,3-dihydro-1-oxo-3-thioxo-2h-isoindo1-2-yl)-pentanedioate(208) and Dimethyl2-(1,3-dihydro-1,3-dithioxo-2H-isoindo1-2-yl)-pentanedioate (209). Amixture of compound 207 (144 mg, 0.47 mmol) and LR (191 mg, 0.47 mmol)in toluene was stirred in a 110° C. oil bath for 10 h. The solvent wasthen evaporated and the residue was purified by column chromatography,(silica gel) using CH₂Cl₂ as the eluent, to obtain compound 209 (17 mg,11%) as a dark red oil. Thereafter, using CH₂Cl₂:EtOAc (10:1) as theeluent the more polar component 208 (105 mg, 70%) was obtained as a redoil.

Compound 208: ¹H NMR (CDCl₃)δ 7.98-7.96 (m, 1H), 7.81-7.70 (m, 3H), 5.53(dd, J=5.1 Hz, J=10 Hz, 1H), 3.70 (s, 3H), 3.59 (s, 3H), 2.76-2.56 (m,2H), 2.40-2.33 (m, 2H); MS (CI/CH₄) m/z 321 (M⁺).

Compound 209: ¹H NMR (CDCl₃)δ 7.87-7.84 (m, 2H), 7.73-7.68 (m. 2H), 6.09(dd, J=5 Hz, J=10 Hz, 1H), 3.70 (s, 3H), 3.58 (s, 3H), 2.81-2.63 (m,2H), 2.40-2.24 (m, 2H); MS (DEI) m/z 337 (M⁺); HRMS (DEI) calcd forC₁₅H₁₅NO₄S₂ 337.0442 (M⁺), found 337.0449.

2-(1,3-Dihydro-1-oxo-3-thioxo-2H-isoindol-2-yl)-pentanedioic acid (2H).Compound 208 (350 mg, 1.09. mmol) was stirred with a 1:1 mixture ofacetic acid glacial and conc. HCl in a 100° C. oil bath for 2.5 h. Ethylacetate (100 mL) and ice water (30 mL) were added. The ethyl acetatelayer was separated, washed with ice water, dried over Na₂SO₄ andconcentrated. The resulting syrup was crystallized with ether to affordcompound 211 as red crystals (253 mg, 79%); mp 157° C.; ¹H NMR(DMSO-d₆)δ 8.04-7.96 (m, 1H), 7.91-7.74 (m, 3H), 5.43 (dd, J=5.1 Hz,J=9.6 Hz, 1H), 2.42-2.33 (m, 2H), 2.30-2.26 (m, 2H); MS (DEI) m/z 293(M⁺); HRMS (DEI) calcd. for C₁₃H₁₁NO₅S 293.0358 (M⁺), found 293.0363;Anal. (C₁₃H₁₁NO₅S) H, N; C: calcd, 53.24; found, 53.88.

2,3-Dihydro-3-thioxo-2-(2,6-dioxo-3-piperidinyl)-1H-isoindol-1-one(212). A mixture of compound 208 (81 mg, 0.276 mmol), trifluoroacetamide(57 mg, 0.50 mmol), 1-hydroxybenzotriazole (145 mg, 1.07 mmol),1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (200 mg,1.04 mmol) and triethylamine (0.21 mL, 1.51 mmol) in CH₂Cl₂ (1.5 mL) wasstirred at ambient for 3 days. Water (10 mL) and CH₂Cl₂ (10 mL) wereadded. The dichloromethane layer was separated, washed with water, driedover Na₂SO₄ and evaporated under reduced pressure. Purification bychromatography, with EtOAc:CH₂Cl₂ (1:10) as the eluent, gave compound212 (48 mg, 63%) as a red solid: mp 255° C.; ¹H NMR (CDCl₃)δ 8.00-7.98(m, 1H), 7.80-7.71 (m, 3H), 5.63 (br, 1H), 2.98-2.70 (m, 3H), 2.18-2.15(m, 1H); MS (CI/CH₄) m/z 274 (M⁺); Anal. (C₁₃H₁₀N₂O₃S) C, H, N.

2-(2, 6-Dithioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione (213) and2,3-dihydro-3-thioxo-2-(2-oxo-6-thioxo-3-piperidinyl)-1H-isoindol-1-one(215). The mixture of 205 (146 mg, 0.533 mmol), LR (108 mg, 0.267 mmol)and pyridine (21 μl) in toluene was stirred at 110° C. under anatmosphere of N₂ for 12 h. Thereafter, additional LR (108 mg, 0.267mmol) and pyridine (21 μl) were added, and the reaction mixture wasstirred for a further 12 h.. The solvent was removed under vacuum andthe residue was purified by column chromatography with CH₂Cl₂:petroleumether (2:1, 10:1) and then CH₂Cl₂:EtOAc (10:1) as eluents to afford 213(30 mg, 45%), 215 (21 mg, 31.5%) and starting material 205 (83 mg).

Compound 213 (yellow solid): mp 263-265° C.; ¹H NMR (CDCl₃)δ 7.78-7.74(m, 2 H), 7.66-7.63 (m, 2 H), 5.00 (dd, J=4.9 Hz, 11.9 Hz, 1 H),3.43-3.35 (m, 1 H), 2.95-2.84 (m, 2 H), 2.08-2.06 (m, 1 H); MS (DEI) m/z290 (M⁺); HRMS (DEI) calcd for C₁₃H₁₀N₂O₂S₂ 290.0184 (M⁺), found290.0185; Anal. (C₁₃H₁₀N₂O₂S₂) C, H, N.

Compound 215 (red solid): mp 240-242° C.; ¹H NMR (CDCl₃)δ 9.44 (s, 1 H),8.05-8.02 (m, 1 H), 7.86-7.76 (m, 3 H), 5.75-5.64 (m, 1 H), 3.57-3.52(m, 1 H), 3.09-2.99 (m, 2 H), 2.19-2.12 (m, 1 H). ¹³C NMR (DMSO):208.16, 207.98, 166.10, 165.39, 134.32, 133.11, 132.42, 124.30, 122.15,121.11, 49.64, 21.29; MS (DEI) m/z 291 (MH⁺); HRMS (DEI) calcd forC₁₃H₁₁N₂O₂S₂ 291.0262 (M⁺), found 291.0264; Anal. (Cl₁₃H₁₀N₂O₂S₂.0.5H₂O)C, H, N.

2,3-Dihydro-3-thioxo-2-(2,6-dithioxo-3-piperidinyl)-1H-isoindol-1-one(216). A mixture of compound 213 (29 mg, 0.1 mmol), LR (22 mg, 0.054mmol) and morpholine (9 μl, 0.1 mmol) in toluene (10 mL) was stirred atreflux under an atmosphere of N₂ for 16 h. The solvent was removed undervacuum and the residue was purified by column chromatography usingCH₂Cl₂:petroleum ether (1:1) as the eluent to afford compound 216 (20mg, 65%) as a red solid: mp 244° C.; ¹H NMR (CDCl₃)δ 10.81 (s, 1H),8.05-8.01 (m, 1H), 7.91-7.75 (m, 3H), 5.92 (m, 1H), 3.57-3.52 (m, 1H),3.13-2.97 (m, 2H), 2.18-2.15 (m, 1H); MS(DEI) m/z 306 (M⁺); HRMS (DEI)calcd for C₁₃H₁₀N₂OS₃ 305.9955 (M⁺), found 305.9951; Anal.(C₁₃H₁₀N₂OS₃.0.5H₂O) C, H, N.

6-Thioxo-2-piperidinone (218). The mixture of glutarimide (0.45 g, 4mmol) and LR (0.809 g, 2 mmol) in THF (30 mL) was stirred at room for 2days. The solvent was evaporated under vacuum and the residue waspurified by column chromatography using petroleum ether:EtOAc (1:1) asthe eluent to give compound 218 as a yellow solid (0.361 g, 70%): mp135° C.; ¹H NMR. (CDCl₃)δ 2.96 (t, J=5.7 Hz, 2 H), 2.58 (t, J=5.8 Hz, 2H), 1.96 (m, 2 H); MS (CI/CH₄) m/z 129 (M⁺); Anal. (C₅H₇NOS) C, H, N.

2,6-Piperidinedithione (219). A mixture of glutarimide (0.34 g, 3 mmol)and LR (1.22 g, 3 mmol) in toluene (30 mL) was stirred at reflux for 3h. The solvent was evaporated under vacuum and the residue was purifiedby column chromatography using petroleum ether:EtOAc (20:1) as theeluent to give compound 219 as a yellow solid (0.286 g, 66%): mp 103°C.; ¹H NMR (CDCl₃)δ 3.02 (t, J=6.3 Hz, 4H), 1.98 (t, J=6.3 Hz, 2H); MS(CI/CH₄) m/z 145 (M⁺); Anal. (C₅H₇NS₂) C, H, N.

2-(3-Cyclohexenyl)-1H-isoindole-1,3(2H)-dione (221). A mixture ofpotassium phthalimide (1.85 g, 3 mmol) and 3-bromocyclohexene (1.79 g, 3mmol) in DMF (15 mL) was stirred in a 100° C. oil bath for 12 h. Thecooled reaction mixture was poured into ice water. The solid wascollected by filtration and purified by flash chromatography with CH₂Cl₂as the eluent to afford compound 221 (1.6 g, 72%) as pink crystals; mp114° C.; ¹H NMR (CDCl₃) δ 7.73-7.69 (m, 2H), 7.62-7.58 (m, 2H),5.85-5.82 (m, 1H), 5.47-5.44 (m, 1H), 4.80-4.78 (m, 1H), 2.14-2.00 (m,3H), 1.86-1.78 (m, 2H), 1.64-1.58 (m, 1H).

2-(3-Cyclohexenyl)-1H-isoindol-1,3(2H)-dithione (222) and2,3-dihydro-3-thioxo-2-(3-cyclohexenyl)-1H-isoindol-1-one (223). Amixture of compound 221 (68 mg, 0.3 mmol) and LR (121 mg, 0.3 mmol) intoluene was refluxed under N₂ for 10 h. The solvent was removed undervacuum and the residue was purified by column chromatography usingpetroleum ether as the eluent to obtain compound 222 (37 mg, 48%) as adark green solid. Then, using CH₂Cl₂:petroleum ether (1:1) as theeluent, the more polar component 223 (23 mg, 32%) was obtained as a redsolid.

Compound 222: mp 93° C.; ¹H NMR (CDCl₃)δ 7.65-7.60 (m, 2H), 7.49-7.42(m, 2H), 5.92-5.88 (m, 1H), 5.66-5.63 (m, 1H), 5.47-5.43 (in, 1H),2.40-2.35 (m, 1H), 1.99-1.95 (m, 2H), 1.75-1.59 (m, 3H); MS (CI/CH₄) m/z259 (M⁺); Anal. (C₁₄H₁₃NS₂) C, H, N.

Compound 223: mp 67-68° C.; ¹H NMR (CDCl₃)δ 7.94- 7.91 (m, 1H),7.73-7.64 (m, 3H), 5.92-5.88 (m, 1H), 5.60-5.51 (m, 2H), 2.27-2.10 (m,3H), 1.96-1.76 (m, 2H), 1.81-1.70 (m, 1H); MS (CI/CH₄) m/z 243 (M⁺);Anal. (C₁₄H₁₃NOS) C, H, N.

Dithiophthalimide (225). A mixture of phthalimide (436 mg, 3.40 mmol)and Lawesson's reagent (1.199 g, 3.40 mmol) in toluene (50 ml) wasrefluxed (oil bath 120° C.) under nitrogen for 5 hours. The solvent wasremoved under vacuum and the residue was directly chromatographed(silica gel, petroleum ether: methylenedichloride/2:3) to givedithiophthalimide as black red needle crystals (240 mg,39.4%):¹HNMR(CDCl₃)δ 9.80 (br, 1H), 7.95 (d, 2H), 7.80 (d, 2H); MS(CI/CH₄) m/z 179 (M⁺).

Example 12 -Synthesis and TNF-α Inhibitory Activity of3-[2′,6′-piperidinedion-3′-yl]-7-amino-2H-1,3-benzoxazine-2,4(3H)-dione

3-[2′, 6′-piperidinedion-3′-yl]-7-amino-2H-1,3-benzoxazine-2,4(3H)-dionewas prepared as shown below in Scheme 17.

4-(t-Butoxycarbonyl amido)salicylic acid (226) was prepared as follows.To a mixture of 4-aminosalicylic acid (306 mg, 2 mmol) and di-t-butyldicarbonate (655 mg, 3 mmol) in H₂O was added NaOH (2N in H₂O) at 0° C.This reaction mixture was allowed to warm to room and then was stirredfor 5 hours. 2N HCl was added dropwise until the mixture wasneutralized. The reaction mixture was then extracted with EtOAc, driedand evaporated to give product (336 mg , 66%) as a dark gray solid:¹HNMR (DMSO-d₆)δ 11.50 (s, 1H), 7.65 (d, 1H), 6.23 (d, 1H), 6.07 (s,1H), 1.70 (s, 9H).

2-[(Ethoxycarbonyl)oxy]-4-(t-butoxycarbonyl amido)-benzoic anhydridewith ethyl hydrogencarbonate (227) was prepared as follows.4-t-Butoxycarbonyl amidosalicylic acid (226) (101 mg, 0.399 mmol) in THF(10 ml) was cooled with dry ice in acetone. Et₃N (0.166 ml) was added,and then ethyl chloroformate (108 mg, 1.135 mmol) was added dropwiseover a period of 30 min. The reaction mixture was stirred at the samefor 5 hours, and then was allowed to warm to room temperature.Thereafter, the reaction mixture was stirred continuously overnight.After evaporation of solvent, the residue was partitioned between waterand ethyl ether. The ether solution was washed with brine, dried overNa₂SO₄ and evaporation of solvent gave product (111 mg, 70%) as a yellowgum: ¹HNMR (CDCl₃)δ 7.75 (d, 1H), 6.48 (d, 1H), 6.38 (s, 1H), 4.38 (m,4H), 1.35 (m, 6H).

3-[2′,6′-piperidinedion-3′-yl]-7-amino-2H-1,3-benzoxazine-2,4(3H)-dione(228) was prepared as follows. A mixture of 227 (32.8 mg, 0.0826 mmol),aminoglutarimide (20 mg, 0.0826 mmol) and Et₃N (25.0 mg, 0.248 mmol in 2ml THF) was stirred at room overnight. Evaporation of solvent gave aresidue which was stirred with a mixture of EtOAc and a saturatedaqueous solution of NaHCO₃. The precipitated white solid was collectedby filtration as the product: ¹HNMR (DMSO-d₆)δ 11.3 (br, 1H), 7.85 (d,1H), 6.80 (d, 1H), 6.60 (s, 1H), 3.15 (t, 2H), 2.15 (t, 2H).

Evaluation of compound 228 in the TNF-α assay described above in Example11 showed that it possessed potent inhibitory action on TNF-α, having anEC₅₀ of 0.4 μM.

Example 13 Angiogenesis Modulating Activity

Angiogenesis is the formation of new blood vessels from pre-existingvessels. Angiogenesis is prominent in solid tumor formation andmetastasis, and is part of the wound healing process. Pathologicalangiogenesis sometimes occurs in inappropriate anatomic locations, suchas the retina or cornea, in response to disease and injury. Inhibitionof angiogenesis could avoid the progression of conditions ofinappropriate angiogenesis.

Tumor formation, for example, requires a network of blood vessels tosustain the nutrient and oxygen supply for continued growth. Tumors inwhich angiogenesis is important include most solid tumors and benigntumors, such as acoustic neuroma, neurofibroma, trachoma, and pyogenicgranulomas. Inhibition of angiogenesis could halt the growth of thesetumors and the resultant damage due to the presence of the tumor.

There is a direct correlation between tumor microvessel density and theincidence of metastasis. Tumor cells themselves can produce factors thatstimulate the proliferation of endothelial cells and new capillarygrowth. Angiogenesis is important in two stages of tumor metastasis. Thefirst stage where angiogenesis stimulation is important is in thevascularization of the primary tumor, which allows tumor cells to enterthe blood stream and to circulate throughout the body. After the tumorcells have left the primary site, and have settled into the secondary,metastatic site, angiogenesis must occur before the metastasis can growand expand. Therefore, inhibiting angiogenesis could lead to thereduction or elimination of metastasis of tumors and possibly containthe neoplastic growth at the primary site. These observations have ledto the investigation of anti-angiogenic agents as possible therapeuticoptions for various cancers.

The angiogenesis modulating activity of representative compounds wasassessed in a rat aortic ring microvessel growth assay. Briefly,twelve-well tissue culture plates were coated with 250 μl of Matrigel(Becton-Dickinson, Bedford, Mass.) and allowed to gel for 30 min at 37°C. and 5% CO₂. Thoracic aortas were excised from 8- to 10-week-old maleSprague Dawley rats. After careful removal of fibroadipose tissues, theaortas were cut into 1-mm-long cross-sections, placed on Matrigel-coatedwells, and covered with an additional 250 μl of Matrigel. After thesecond layer of Matrigel had set, the rings were covered with EGM-II andincubated overnight at 37° C. and 5% CO2. EGM-II consists of endothelialcell basal medium (EBM-II; Clonetics, San Diego, Calif.) plusendothelial cell growth factors provided as the EGM-II Bulletkit(Clonetics). The culture medium was subsequently changed to EBM-IIsupplemented with 2% fetal bovine serum, 0.25 μg/ml amphotericin B, and10 μg/ml gentamicin. Aortic rings were treated daily with either thevehicle (0.5% DMSO), carboxyamidotriazole (CAI, 12 μg/ml), thalidomideor thalidomide analogs (0.1-20 μg/ml) for 4 days and photographed on the5th day using a ×2.5 objective. CAI, a known antiangiogenic agent, wasused at higher than clinically achievable concentration as a positivecontrol. Experiments were repeated four times using aortas from fourdifferent rats. The area of angiogenic sprouting, reported in squarepixels, was quantified using Adobe PhotoShop. Further details of themethod are provided in Luzzio et al., J Med Chem.; 46:3793-9, 2003,which is incorporated by reference herein. It should be understood thatthis method can be used as an assay to rapidly select compounds having adesired angiogenic or anti-angiogenic effect, for example, for use inthe disclosed methods of treating a subject.

Bar graphs showing the results of the angiogenesis assay for severalcompounds are shown in FIGS. 2-11. For convenience, the structures ofthe assayed compounds also are presented in these figures.

FIG. 2 shows the angiogenic modulating activity of1,3-Dioxo-2-(2-hydroxy-6-methoxypyridin-3-yl)-isoindoline hydrobromideat several concentrations. This compound exhibited anti-angiogenicactivity in the rat aortic ring assay at all concentrations tested.

FIG. 3 shows the angiogenic modulating activity of2-(3-cyclohexenyl)-H-isoindol-1,3(2H)-dithione at severalconcentrations. This compounds exhibited anti-angiogenic activity athigher concentrations and angiogenic activity at lower concentrations.

FIG. 4 shows the angiogenic modulating activity of1-(2,6-Dithioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione at severalconcentrations. This compound exhibited anti-angiogenic activity at allconcentrations tested.

FIG. 5 shows the angiogenic modulating activity of 3-Camphanicamino-2,6-piperidinedione at several concentrations. This compoundexhibited potent angiogenic activity at all concentrations tested,making this compound promising for treating conditions where increasedangiogenesis is desired, for example, as an aid to wound healing.

FIG. 6 shows the angiogenic modulating activity of Dithiophthalimide atseveral concentrations. This compound exhibited angiogenic activity atall concentrations tested.

FIG. 7 shows the angiogenic modulating activity of2-(1,3-Dihydro-1-oxo-3-thioxo-2H-isoindol-2-yl)-pentanedioic acid atseveral concentrations. This compound exhibited angiogenic activity atall concentrations tested.

FIG. 8 shows the angiogenic modulating activity of2-(2-Oxo-6-thioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione at severalconcentrations. This compound showed anti-angiogenic activity at higherconcentration, and some angiogenic activity at lower concentrations.

FIG. 9 shows the angiogenic modulating activity of2,3-Dihydro-3-thioxo-2-(2,6-dithioxo-3-piperidinyl)-1H-isoindol-1-one atseveral concentrations. This compound exhibited potent anti-antigiogenicactivity at higher concentrations and angiogenic activity at lowerconcentrations.

FIG. 10 shows the angiogenic modulating activity of2-Acetoxy-N-(2,6-dioxopiperidin-3-yl)benzamide at severalconcentrations. At all concentrations tested, this compound exhibitedpotent angiogenic activity.

FIG. 11 shows the angiogenic modulating activity of1,3-Dioxo-2-(2,6-dimethoxypyridin-3-yl)-isoindoline at severalconcentrations. This compound exhibited angiogenic activity at allconcentrations tested.

In summary, the disclosed compounds exhibit a range of angiogenicmodulating activities ranging from potent inhibition of angiogenesis(anti-angiogenic activity) to potent stimulation of angiogenesis(angiogenic activity). Some compounds exhibit both angiogenic andanti-angiogenic activity in a dose-dependent manner. Those compounds (orparticular concentrations thereof) having angiogenic activity are usefulfor treating conditions or diseases where increasing angiogenesis isdesirable (for example, wound healing) and those compounds (orparticular concentrations thereof) having anti-angiogenic activity areuseful for treating conditions or diseases where decreasingangiongenesis is desirable (for example, cancers, diabetic retinopathyor corneal neovascularization). Persons of ordinary skill in the art canuse the assay described above (or other known angiogenic/anti-angiogenicactivity assays) to readily determine amounts of the disclosed compoundsthat therapeutically effective for stimulating or inhibitingangiogenesis as appropriate for a given subject's condition.

Example 14 Synthesis of 3-Camphanic amino-2,6-piperidinedione

A mixture of (+)-camphanic chloride (19 mg, 00868 mmol),aminoglutarimide (21 mg, 0.0868 mmol) and Et₃N (24 μl ) in CHCl₃ (1 ml)was stirred at room for 16 hours. The solution was diluted with CHCl₃,washed with saturated aqueous solution of NaHCO₃, dried over Na₂SO₄,concentrated and purified by chromatography (silica gel, CH2C12 ;EtOAc=10:1) to give product (16 mg, 60.0% yield) as a colorless gel:¹³CNMR (CDCl₃)δ 172.6, 169.4, 168.6; 165.5, 90.3, 58.3, 53.3, 48.2,47.6, 29.2, 28.2, 26.9, 22.7, 14.6, 7.6; MS (CI/CH₄) m/z 308 (M⁺). Thiscompound exhibited angiogenic activity in the assay of Example 13.

Example 15 Synthesis of 3-Benzylimino-2-benzyl-2,3-dihydroisoindol-1-one

A solution of Dimethyl2-(1,3-dihydro-1-oxo-3-thioxo-2H-isoindo1-2-yl)-pentanedioate (compound208 of Example 11, 100mg, 0.311 mmol) and benzylamine was stirred in a50° C. oil bath for 5 hours. The reaction mixture was partitionedbetween water and ethyl acetate. The organic layer was washed withwater, dried, and concentrated. The residue was purified bychromatography (silica gel, CH₂Cl₂) to give product as white crystals(60 mg, 59.0%): ¹HNMR (CDCl₃) δ 7.10-7.90 (m, 10 H), 5.18 (s, 2H), 4.95(s, 2H);¹³CNMR(CDCl₃)δ 167.8, 151.3, 140.6, 138.2, 133.5, 133.3, 132.1,130.4, 130.1, 129.1, 128.8, 128.7, 128.5, 128.4, 127.9, 127.6, 127.5,127.2, 126.0, 124.1, 53.9, 42.5; FAB-MS m/z 327 (MH⁺). This compoundexhibited angiogenic activity in the assay of Example 13.

Example 16 Thionation

Although many of the disclosed compounds are illustrated without thionylgroups in their structures, it is to be understood that any of thecarbonyl groups shown in the structures of the disclosed compounds maybe converted into thiocarbonyl groups, and that such thio-derivativesare part of this disclosure. Thionation may be accomplished by any knownmethod. Particular methods of thionation include use of phosphoruspentasulfide, hydrogen sulfide, O,O-diethyldithiophosphonic acid, boronsulfide, silicon disulfide and elemental sulfur in HMPA. However, aparticularly convenient method of thionation is the use of2,4-bis(p-methoxyphenyl)-1,3-dithiadiphosphetane-2,4-disulfide and itsderivatives (generically “Lawesson's Reagents”). These reagents aredescribed in Cava and Levinson, “Thionation Reactions of Lawesson'sReagents,” Tetrahedron, 41: 5061-5087, 1985, which is incorporated byreference herein.

Example 17 Pharmaceutical Compositions

The disclosed pharmaceutical compositions can be in the form of tablets,capsules, powders, granules, lozenges, liquid or gel preparations, suchas oral, topical, or sterile parenteral solutions or suspensions (e.g.,eye or ear drops, throat or nasal sprays, etc.), transdermal patches,and other forms known in the art.

Pharmaceutical compositions can be administered systemically or locallyin any manner appropriate to the treatment of a given condition,including orally, parenterally, rectally, nasally, buccally, vaginally,topically, optically, by inhalation spray, or via an implantedreservoir. The term “parenterally” as used herein includes, but is notlimited to subcutaneous, intravenous, intramuscular, intrasternal,intrasynovial, intrathecal, intrahepatic, intralesional, andintracranial administration, for example, by injection or infusion. Fortreatment of the central nervous system, the pharmaceutical compositionsmay readily penetrate the blood-brain bather when peripherally orintraventricularly administered.

Pharmaceutically acceptable carriers include, but are not limited to,ion exchangers, alumina, aluminum stearate, lecithin, serum proteins(such as human serum albumin), buffers (such as phosphates), glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol, andwool fat.

Tablets and capsules for oral administration can be in a form suitablefor unit dose presentation and can contain conventional pharmaceuticallyacceptable excipients. Examples of these include binding agents such assyrup, acacia, gelatin, sorbitol, tragacanth, and polyvinylpyrrolidone;fillers such as lactose, sugar, corn starch, calcium phosphate,sorbitol, or glycine; tableting lubricants, such as magnesium stearate,talc, polyethylene glycol, or silica; disintegrants, such as potatostarch; and dispersing or wetting agents, such as sodium lauryl sulfate.Oral liquid preparations can be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or can bepresented as a dry product for reconstitution with water or othersuitable vehicle before use.

The pharmaceutical compositions can also be administered parenterally ina sterile aqueous or oleaginous medium. The composition can be dissolvedor suspended in a non-toxic parenterally-acceptable diluent or solvent,e.g., as a solution in 1,3-butanediol. Commonly used vehicles andsolvents include water, physiological saline, Hank's solution, Ringer'ssolution, and sterile, fixed oils, including synthetic mono- ordi-glycerides, etc. For topical application, the drug may be made upinto a solution, suspension, cream, lotion, or ointment in a suitableaqueous or non-aqueous vehicle. Additives may also be included, forexample, buffers such as sodium metabisulphite or disodium edeate;preservatives such as bactericidal and fungicidal agents, includingphenyl mercuric acetate or nitrate, benzalkonium chloride orchlorhexidine, and thickening agents, such as hypromellose.

The dosage unit involved depends, for example, on the condition treated,nature of the formulation, nature of the condition, embodiment of theclaimed pharmaceutical compositions, mode of administration, andcondition and weight of the patient. Dosage levels are typicallysufficient to achieve a tissue concentration at the site of action thatis at least the same as a concentration that has been shown to be activein vitro, in vivo, or in tissue culture. For example, a dosage of about0.1 μg/kg body weight/day to about 1000 mg/kg body weight/day, forexample, a dosage of about 1 μg/kg body weight/day to about 1000 μg/kgbody weight/day, such as a dosage of about 5 μg/kg body weight/day toabout 500 μg/kg body weight/day can be useful for treatment of aparticular condition.

The compounds can be used in the form of pharmaceutically acceptablesalts derived from inorganic or organic acids and bases, including, butnot limited to: acetate, adipate, alginate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, citrate, camphorate,camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.Base salts include, but are not limited to, ammonium salts, alkali metalsalts (such as sodium and potassium salts), alkaline earth metal salts(such as calcium and magnesium salts), salts with organic bases (such asdicyclohexylamine salts), N-methyl-D-glucamine, and salts with aminoacids (such as arginine, lysine, etc.). Basic nitrogen-containing groupscan be quaternized, for example, with such agents as C1-8 alkyl halides(such as methyl, ethyl, propyl, and butyl chlorides, bromides, andiodides), dialkyl sulfates (such as dimethyl, diethyl, dibutyl, andiamyl sulfates), long-chain halides (such as decyl, lauryl, myristyl,and stearyl chlorides, bromides, and iodides), aralkyl halides (such asbenzyl and phenethyl bromides), etc. Water or oil-soluble or dispersibleproducts are produced thereby.

Pharmaceutical compositions can be included in a kit accompanied byinstructions for intended use, for example instructions required by apharmaceutical regulatory agency, such as the Food and DrugAdministration in the United States.

In view of the many possible embodiments of the invention that exist, itshould be recognized that the illustrated embodiments are only examplesof the invention and should not be taken as a limitation on the scope ofthe invention. Rather, the scope of the invention is defined by thefollowing claims. We therefore claim as our invention, all that comeswithin the scope and spirit of these claims.

1-56. (canceled)
 57. A composition comprising Compound I and apharmaceutically acceptable excipient.


58. A method of treating an inflammatory disorder comprisingadministering the composition of claim
 57. 59. The method of claim 58wherein said inflammatory disorder is selected from low-gradeinflammation associated obesity and obesity-related co-morbidities. 60.The method of claim 58 wherein said inflammatory disorder is anarthritic condition.
 61. The method of claim 60 wherein said arthriticcondition is selected from rheumatoid arthritis, rheumatoid spondylitis,and osteoarthritis.
 62. The method of claim 58 wherein said inflammatorydisorder is sepsis.
 63. The method of claim 58 wherein said inflammatorydisorder is bacterial meningitis.
 64. The method of claim 58 whereinsaid inflammatory disorder is selected from inflammatory bowel disease,Crohn's Disease and ulcerative colitis.
 65. The method of claim 58wherein said inflammatory disorder comprises an inflammation-associatedcognitive dysfunction.
 66. A method of modulating TNF-alpha activity inan individual, the method comprising administering to said individual aneffective amount of the composition according to claim
 57. 67. A methodof treating a neuroinflammatory conditions comprising the step ofadministering to an individual the composition of claim
 57. 68. Themethod of claim 67 wherein said neuroinflammatory condition comprisesAlzheimer's Disease,
 69. The method of claim 67 wherein saidneuroinflammatory condition comprises Parkinson's disease.
 70. Themethod of claim 67 wherein said neuroinflammatory condition comprisesfrontotemporal dementia.
 71. The method of claim 67 wherein saidneuroinflammatory condition comprises a motor neuron disease.
 72. Themethod of claim 71 wherein said motor neuron disease comprisesamyotrophic lateral sclerosis.
 73. The method of claim 67 wherein saidneuroinflammatory condition comprises a CNS injury.
 74. The method ofclaim 67 wherein said neuroinflammatory condition comprises strokeand/or hemorrhage.
 75. The method of claim 67 wherein saidneuroinflammatory condition comprises multiple sclerosis.
 76. A methodof treating an inflammation-associated ocular disease comprising thestep of administering the composition of claim
 57. 77. The method ofclaim 76, wherein said ocular disease is selected from uveitis andmacular degeneration.
 78. A method of treating angiogenesis and/or tumorgrowth in cancer, comprising the step of administering the compositionof claim 57.